Magnetic immobilized lipase and application thereof in resolution of 1-methyl-3-amphetamine

A technology of immobilizing lipase and lipase, applied in the direction of immobilized lipase, lipase, enzyme, hydrolase, etc. on or in inorganic carrier, can solve the problems of reduced conversion efficiency, poor water solubility of substrate, restriction enzyme application, etc. High, splitting ability, the effect of optimizing immobilization conditions

Active Publication Date: 2020-04-21
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymatic separation of chiral compounds has important application prospects, but in the process of biotransformation, the problem of poo

Method used

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  • Magnetic immobilized lipase and application thereof in resolution of 1-methyl-3-amphetamine
  • Magnetic immobilized lipase and application thereof in resolution of 1-methyl-3-amphetamine
  • Magnetic immobilized lipase and application thereof in resolution of 1-methyl-3-amphetamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Screening of free lipase

[0034] Take 100 mg of 12 kinds of lipases from different manufacturers (numbered for convenience to distinguish different manufacturers) into 10 mL reaction bottles of the same specification, respectively add solvent anhydrous toluene 1800 μL, acylating reagent ethyl acetate 200 μL, substrate 60 μL of 1-methyl-3-amphetamine was reacted in a shaker at 40°C and 150 rpm for 5 days. After the reaction, the enzyme was centrifuged from the reaction solution, and the organic solvent was removed by a rotary evaporator to obtain a transparent crystal. Add 2 mL of iso Dissolve propanol, filter with an organic filter head with a pore size of 0.45 μm, and analyze (S)-1-methyl-3-amphetamine and (R)-(+)-N-acetyl-1-methyl- 3-amphetamine content, determination of substrate conversion and enantiomeric excess of (R)-(+)-N-acetyl-1-methyl-3-amphetamine. The results are shown in Table 1. The results show that the lipase LIP10 has the best enantiose...

Embodiment 2

[0037] Example 2: Preparation of Magnetically Immobilized Lipase

[0038] (1) Fe 3 o 4 Magnetic nanoparticles: 5.6g FeSO 4 ·7H 2 O and 10 g FeCl 3 ·6H 2 O was respectively dissolved in 40mL distilled water, mixed and put into a 250mL three-neck flask, 150mL deionized water was added, stirred at a high speed at 1000r / min and 40°C, and 40mL ammonia water (NH 3 ·H 2 O solution), when the solution becomes black and bright and the pH of the supernatant is ≥ 10.0, adjust the temperature to 60°C and continue stirring for 30min, then adjust the temperature to 70°C and stir for 1h. After the reaction is over, use absolute ethanol and Repeated washing with deionized water 10 times until the supernatant was neutral (i.e. pH 7.0), recovered by a magnet, frozen in an ultra-low temperature -80°C refrigerator for 7 hours, and then freeze-dried at -60°C for 12 hours to obtain Fe 3 o 4 Magnetic nanoparticles 2.8g, particle size 12-30nm.

[0039] (2) APTES-Fe 3 o 4 Magnetic nanoparti...

Embodiment 3

[0042] Embodiment 3: the influence of reaction time on the selective acylation of 1-methyl-3-amphetamine

[0043]In six 10ml reaction flasks, add 1800 μL of solvent anhydrous toluene, 200 μL of acylating reagent ethyl acetate, 60 μL of substrate 1-methyl-3-amphetamine, and 100 mg of magnetically immobilized lipase prepared by the method in Example 2, respectively. 1d, 2d, 3d, 4d, 5d, 6d were respectively reacted in a shaker at 40°C and 150rpm. After the reaction, use an external magnetic field to recover the precipitate, separate the enzyme from the reaction solution, and use a rotary evaporator to remove the organic solvent to obtain a transparent crystal, add 2 mL of isopropanol, filter with an organic filter head with a pore size of 0.45 μm, and analyze by HPLC The content of (S)-1-methyl-3-amphetamine and (R)-(+)-N-acetyl-1-methyl-3-amphetamine in the sample determines the substrate conversion rate and (R)-( +)-N-acetyl-1-methyl-3-amphetamine enantiomeric excess, the resu...

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Abstract

The invention discloses a magnetic immobilized lipase and an application thereof in preparation of (R)-(+)-N-acetyl-1-methyl-3-amphetamine through resolution of 1-methyl-3-amphetamine. According to the method, glutaraldehyde is replaced with carbon disulfide, dithiocarbamate is introduced to the surfaces of magnetic particles to be covalently combined with lipase. The method for preparing the magnetic immobilized lipase is simple and safe and is obviously superior to a glutaraldehyde cross-linking enzyme immobilization method. Meanwhile, the magnetic immobilized lipase is applied to an alternating magnetic field to catalyze an asymmetric acylation reaction, so that continuous preparation of (R)-(+)-N-acetyl-1-methyl-3-amphetamine is realized. The method is simple, safe, effective and easyin resolution. The enantiomer excess value of an R-configuration product obtained by resolution of 1-methyl-3-amphetamine reaches 98.5%, and the resolution effect is excellent.

Description

[0001] (1) Technical field [0002] The invention relates to a preparation method of (R)-(+)-N-acetyl-1-methyl-3-amphetamine. In particular, it relates to a method for preparing (R)-(+)-N-acetyl-1-methyl-3-amphetamine by resolving racemic 1-methyl-3-amphetamine with magnetically immobilized enzyme . [0003] (2) Background technology [0004] (R)-(+)-N-acetyl-1-methyl-3-amphetamine (N-[(2R)-4-phenylbutan-2-yl]acetamide), CAS22148-79-4, molecular formula C 12 h 16 NO, molecular weight 191.27, melting point 55°C, boiling point 360.5±21°C. It is a key chiral intermediate in the synthesis of the antihypertensive drug labetalol. Labetalol, also known as benzalate, is an ideal antihypertensive drug for emergency treatment of essential hypertension and secondary hypertension, hypertension during pregnancy and other types of hypertension. The labetalol key chiral intermediate (R)-(+)-N-acetyl-1-methyl-3-amphetamine can be prepared by splitting 1-methyl-3-amphetamine with lipase. ...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N11/14C12P41/00C12P13/00
CPCC12N11/14C12N9/20C12Y301/01003C12P41/00C12P13/001
Inventor 欧志敏代洪倩柳博卢媛唐岚杜理华
Owner ZHEJIANG UNIV OF TECH
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