Preparation and detection of 9-Fluorenmethoxycarbonylsuccinimide_Industrial additives

Preparation and detection background and overview of 9-fluorenemethoxycarbonylsuccinimide

9-Fluorenmethoxycarbonylsuccinimide (referred to as Fmoc-ONSu) is a colorless or light yellow needle-shaped crystal with a molecular formula of C19H15NO5 and a molecular weight of 337.3. 9-Fluorenmethoxycarbonylsuccinimide is mainly It is used to protect the processing of amino acids in products used for peptide synthesis. It is an organic compound between amino acids and proteins. Through research on the synthesis process of peptides, it has become one of the important research directions in the current field of biochemistry.

Preparation and detection preparation of 9-fluorenemethoxycarbonylsuccinimide

Existing peptide synthesis generally adopts solid-phase or synthetic methods. On the one hand, raw materials are consumed a lot during synthesis and the synthesis equipment is expensive. The processing and synthesis efficiency is low, the purity is low, and the synthesis reaction process is complicated and difficult to separate; On the one hand, the equipment used for synthesis is expensive and the production cost is high, which cannot meet the needs of small and medium-sized enterprises for processing and synthesis. Therefore, based on the above problems, CN201310271359.8 provides a method for preparing 9-fluorenylmethoxycarbonylsuccinimide.

The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a method for preparing 9-fluorenylmethoxycarbonylsuccinimide, which can greatly improve the synthesis production efficiency and the purity of the synthetic products, and on the other hand, can greatly improve the synthesis production efficiency and the purity of the synthetic products. On the one hand, the equipment used for synthesis is moderately priced and can meet the requirements of small and medium-sized enterprises.

Technical solution: The present invention provides a method for preparing 9-fluorenemethoxycarbonylsuccinimide, which includes the following steps,

Step 1. First, mix fluorene methoxycarbonyl chloride formate and imine succinate aqueous solution with a mass ratio of 1:3-4

As raw materials, then control the reaction temperature at 40℃-60℃, slowly stir the reaction for 10m-30m to obtain the reaction liquid;

Step 2. Add an inorganic base to the reaction solution in step 1 and stir the reaction. The inorganic base is sodium carbonate or sodium bicarbonate, and the mass ratio is 2-4 times that of imine succinate aqueous solution;

Step 3: Filter, wash, and dry the reaction solution prepared in Step 3 to obtain 9-fluorenylmethoxycarbonylsuccinimide, where the drying temperature is 30°C-80°C.

The preferred concentration of the fluorenemethoxycarbonyl chloride formate solution in step 1 is 7%-10%.

The preferred concentration of imine succinate aqueous solution in step 1 is 6%-9%.

The preferred reaction temperature in step 1 is 45°C-55°C, and the reaction time is 15 m-20 m.

Preferably, the inorganic base in step 2 is sodium carbonate or sodium bicarbonate, with a mass ratio of 2.5-3 times that of the aqueous immonium succinate solution.

The preferred drying temperature in step 3 is 50°C-60°C.

Compared with the existing technology, the beneficial effects of the present invention are:

The present invention is a method for preparing 9-fluorenylmethoxycarbonylsuccinimide, which solves the existing problems of low processing and synthesis efficiency, low purity and complicated methods, and can be processed and synthesized quickly and simply. High-purity 9-Fluorenmethoxycarbonylsuccinimide: The equipment used for simultaneous processing and synthesis is moderately priced, which meets the processing and synthesis needs of small and medium-sized enterprises and reduces enterprise production cost investment.

Preparation and detection method of 9-fluorenemethoxycarbonylsuccinimide

9-Fluorenmethoxycarbonylsuccinimide is an important raw material for organic synthesis and is currently mainly used as an intermediate for peptide synthesis and anti-thrombotic drugs. Currently, there are many research reports on the synthesis and application of Fmoc-ONSu, but there is no published information on the analysis method of Fmoc-ONSu. Although there are literatures or patents that mention the liquid chromatography content of Fmoc-ONSu synthesis products, the detailed operating conditions of liquid chromatography have not been disclosed. What type of high-performance liquid chromatography column is used and the specific operating conditions need to be detailed. groping.

The commonly used method at present is to perform pre-column or post-column derivatization, that is, the sample first reacts with the derivatization reagent to generate a stable derivatization product with UV chromophore or fluorophore, and then obtains it through UV light or fluorescence detector. Corresponding response, so that the actual content of the sample can be detected. Although this method can theoretically be used to determine the true content of Fmoc-ONSu, it has major drawbacks due to the scarcity of derivatizing agents and cumbersome operating procedures.

In order to overcome the shortcomings of the existing technology, provide an easy-to-operate, test�Accurate liquid chromatography detection method. The present invention discloses a liquid chromatography method for determining the purity of 9-fluorenylmethoxycarbonylsuccinimide.

The present invention achieves the purpose of the invention through the following technical solutions:

A liquid chromatography method for determining the purity of 9-fluorenethoxycarbonylsuccinimide nickel amine, which is operated by a high-performance liquid chromatograph, and is characterized by: following the following method in sequence:

a. Set up a high-performance liquid chromatograph: The chromatographic column of the high-performance liquid chromatograph is used as the stationary phase, and the stationary phase is a reversed-phase bonded stationary phase. That is, the filler of the chromatographic column is selected from octadecyl bonded phase silica gel, octyl bonded phase Either alkyl bonded phase silica gel or cyano bonded phase silica gel, the particle size of the filler is not greater than 5 μm, the length of the chromatographic column is 10cm~50cm, and the inner diameter of the chromatographic column is 4mm~5mm;

The mobile phase of high performance liquid chromatography can be any one of water, acetonitrile, tetrahydrofuran, cyclohexane, n-hexane, diethyl ether, dichloromethane, chloroform and toluene;

The detector of the high-performance liquid chromatograph adopts a multi-wavelength variable UV-visible light detector, and the wavelength of the ultraviolet emitted by the detector is between 220nm and 370nm;

b. Prepare standard sample solution: Use chromatographically pure mobile phase as solvent, and use a volumetric flask or other instruments to prepare the standard sample of 9-fluorenylmethoxycarbonylsuccinimide to a concentration of 0.1 mg/mL ~ 1.2 mg. /mL solution is used as the standard sample for analysis. When preparing, use ultrasonic degassing and dissolution, mix and shake the solution well, and use it as the standard sample for analysis;

c. Prepare the sample solution to be tested: Use a chromatographically pure mobile phase as the solvent, and use a volumetric flask or other instruments to prepare the sample to be tested containing 9-fluorenylmethoxycarbonylsuccinimide to a concentration of 0.1 mg/mL. The solution of ~1.2 mg/mL is used as the sample to be analyzed. When preparing, use ultrasonic degassing and dissolution, mix and shake the solution well, and use it as the standard sample for analysis;

d. Analyze standard samples: After the conditions of the HPLC are stable, use the HPLC to analyze the standard samples. Use a 50 μL micro-syringe to inject the standard samples into the HPLC. Each group of standard samples After the components are separated, the peak area of ​​the tri-tert-butylphosphine tetrafluoroborate component of the 9-fluorenemethoxycarbonylsuccinimide component in the standard sample is obtained;

e. Analyze the sample: After the conditions of the HPLC are stable, use the HPLC to analyze the sample, and use a 50 μL micro-syringe to inject the sample into the HPLC. After the components are separated, the peak area of ​​the 9-fluorenemethoxycarbonylsuccinimide component in the sample is obtained;

f. Calculate the purity: Use the external standard method to quantify the purity of the sample with the standard sample. The purity of 9-fluorenylmethoxycarbonylsuccinimide in the sample to be tested is expressed in mass fraction and calculated according to the following formula:

X1: The purity of 9-fluorenemethoxycarbonylsuccinimide in the sample, expressed in percentage,

X0: The purity of 9-fluorenemethoxycarbonylsuccinimide in the standard sample, expressed as a percentage,

A1: Peak area of ​​9-fluorenemethoxycarbonylsuccinimide component in the sample,

A0: Peak area of ​​9-fluorenemethoxycarbonylsuccinimide component in the standard sample,

m1: mass of the sample,

m0: The quality of the standard sample.

The analysis process using the external standard method is fast, convenient and easy to operate. The error of the analysis results is small. The obtained chromatographic peaks have sharp peak shapes and good symmetry. It can be used as the synthesis and purification process of 9-fluorenylmethoxycarbonylsuccinimide. The purity test is suitable for 9-fluorenemethoxycarbonylsuccinimide samples of various purity.