Synthesis method of enzyme-catalyzed (1S, 5R)-bicyclolactone

A synthesis method and bicyclic lactone technology are applied in the field of biopharmaceuticals, which can solve the problems of small production scale and troublesome post-processing, and achieve the effects of improving separation conditions and avoiding the use of chemical catalytic reagents.

Pending Publication Date: 2020-08-25
FUDAN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

However, these methods have shortcomings such as small production scale and troublesome post-processing.

Method used

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  • Synthesis method of enzyme-catalyzed (1S, 5R)-bicyclolactone
  • Synthesis method of enzyme-catalyzed (1S, 5R)-bicyclolactone
  • Synthesis method of enzyme-catalyzed (1S, 5R)-bicyclolactone

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, construction and induced expression of genetically engineered bacteria

[0043] Transform the expression host BL21(DE3) with the plasmid CHMO-Rhodo1. Inoculate BL21(DE3)-CHMO-Rhodo1 into a 5mL liquid LB test tube culture medium containing kanamycin resistance, and activate it on a shaker at 37°C for 8 hours , the activated culture was transferred to the liquid LB shake flask medium containing kanamycin resistance according to 1% transfer amount, and the fermentation medium was shaken at constant temperature for 3 hours, and the culture temperature was 37°C, 200rpm. When the cell concentration grows to OD 600 When = 1.2, add 0.1mM IPTG (final concentration), induce at 18°C ​​for 18h, collect cells by centrifugation at 8500rpm for 10min, resuspend with pH7.5, 250mM sodium phosphate buffer, break with 45% ultrasonic power for 30min, and centrifuge to obtain The cell supernatant containing CHMO-Rhodo1 was stored in a 4-degree refrigerator.

Embodiment 2

[0044] Embodiment 2, reaction process monitoring

[0045] Adopt GC detection method to measure racemic substitution bicyclo [3.2.0]-hept-2-en-6-ketone (II) to (1 S , 5 R )-transformation of bicyclic lactone (I). Take 100 L of the reaction solution at different reaction time points, add an equal volume of ethyl acetate solution, mix well, centrifuge to take the upper ethyl acetate layer, and filter it with a 0.3M filter membrane for detection. Chromatographic conditions: chromatographic column DB-624 (0.32mm × 30m), detector: DAD detector, sample volume: 1 L, heating program: initial temperature: 80°C, 80-130°C: heating rate 10°C / min, 130-230°C: heating rate 20°C / min, hold for 2min. The retention time of racemic substituted bicyclo[3.2.0]-hept-2-en-6-one (II) is 7.8min, and its structure identification 1 H NMR as Figure 5 As shown, (1 S , 5 R )-bicyclic lactone (I) has a retention time of 11.02min, and its structure identification 1 H NMR as Image 6 shown. The absol...

Embodiment 3

[0046] Embodiment 3, the mensuration of product ee value

[0047] target product (1 S , 5 R )-The determination of the ee value of bicyclic lactone (I) adopts the method of chiral SFC to measure. Sample treatment: After the post-treatment product was dissolved in chromatographic grade anhydrous methanol, it was detected by SFC.

[0048] SFC analysis conditions: Chromatographic column: Chiralpak IF (4.6mm × 250mm), mobile phase: CO 2 / MeOH=95:5, flow rate: 3mL / min, injection volume: 5 L, detection wavelength: 220nM, the HPLC spectrum of the racemic product is as follows figure 2 As shown, (1R, 5 S )-7, the retention time of 7-dichlorobicyclic lactone is 2.82min, (1 S , 5 R )-7,7-dichlorobicyclic lactone (I) has a retention time of 3.029min.

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Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a synthesis method of enzyme-catalyzed (1S, 5R)-bicyclolactone. The method comprises the following steps of preparing engineering bacteria containing Bayer-Villiger monooxidase; preparing a resting cell suspension of the engineering bacteria, ultrasonically crushing or pressurizing and crushing to obtain a supernatant containing Bayer-Villiger monooxidase cells, mixing the supernatant with a substrate racemic substituted bicyclo [3.2.0]-hept-2-ene-6-one, a hydrogen donor and a cofactor, and carrying out asymmetric Bayer-Villiger oxidation to prepare (1S, 5R)-bicyclolactone, wherein an amino acid sequence of the Bayer-Villiger oxidase is shown as SEQ ID NO. 1. According to the method, racemic substituted bicyclo [3.2.0]-hept-2-ene-6-one (II) can be catalyzed to generate chiral (1S, 5R)-bicyclolactone, the used engineering bacteria is convenient and easy to obtain, and the product is convenient to separate and high in enantioselectivity.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to enzyme-catalyzed (1 S , 5 R )-the synthetic method of bicyclic lactone (I). Background technique [0002] Known as shown in formula (I) (1 S , 5 R )-bicyclic lactone is a key intermediate in the synthesis of prostaglandins: [0003] [0004] In the formula, R is hydrogen, fluorine, chlorine, bromine, iodine and other halogens, C 1 -C 8 Alkyl or cycloalkyl, phenyl, monosubstituted or polysubstituted aryl or aralkyl, thienyl, furyl, naphthyl, pyridyl, etc. n is 1~10. [0005] Its synthesis was first made by Tolstikov, G. A. et al. ( Zhurnal Organicheskoi Khimii , 1989, 25 , 208) reported that the racemic substrate was synthesized mainly from cyclopentadiene in three steps, followed by diastereomeric crystallization resolution with (R)-(+)-methylbenzylamine, and internal Esterification affords the desired (1 S , 5 R )-bicyclic lactone (I). This method has ...

Claims

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Application Information

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IPC IPC(8): C12P17/04C12N15/70C12N9/02C12N9/04C12R1/19
CPCC12P17/04C12N15/70C12N9/0073C12N9/0006C12Y114/13022C12Y101/9901C12N1/20
Inventor 陈芬儿竺科杰黄则度程荡王佳琦陶媛
Owner FUDAN UNIV
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