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New method for using controllable multi-enzyme cascade reaction to synthesize optical pure allylic epoxy ketone or alcohol

An epoxy ketone, optical technology, applied in microorganism-based methods, biochemical equipment and methods, introduction of foreign genetic material using carriers, etc., to achieve the effects of easy operation, high ee and de values, and simple reaction system

Active Publication Date: 2015-11-25
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no biological method reported to synthesize optically pure epoxy ketones by asymmetric epoxidation of α,β-unsaturated ketones

Method used

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  • New method for using controllable multi-enzyme cascade reaction to synthesize optical pure allylic epoxy ketone or alcohol
  • New method for using controllable multi-enzyme cascade reaction to synthesize optical pure allylic epoxy ketone or alcohol
  • New method for using controllable multi-enzyme cascade reaction to synthesize optical pure allylic epoxy ketone or alcohol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of biocatalyst E.coliBL21ΔnemA (pRSFD-REAB)

[0028] (1) Construction method of Escherichia coli expression strain E.coliBL21ΔnemA

[0029] Since the N-acetylmaleimide reductase expressed by the nemA gene in the E.coliBL21 genome can efficiently reduce the C=C bond of the substrate ɑ,β-unsaturated ketone, we introduced the λRed recombination and knocked out the E.coliBL21 genome The nemA gene.

[0030] Using P1 / P3 and P4 / P2 as primers (Table 1) and E. coliBL21 genomic DNA as a template, homologous sequences on both sides of nemA gene were amplified respectively. PCR amplification conditions: pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 0.5min, 30 cycles, and final extension at 72°C for 10min.

[0031] Using FRTf and FRTr as primers (Table 1) and plasmid pKD4 as a template, the kanamycin resistance gene fragment was amplified. Due to the different lengths of the fragments duri...

Embodiment 2

[0043] Example 2: Synthesis of (3R,4S)-4-phenyl-3,4-epoxy-2-butanone

[0044] Take 1.5 g of the bacteria from Example 1 and resuspend in 10 ml potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer (0.1M, pH=7.0), add 20 mM model substrate 4-phenyl-3-butene-2 - Ketone (1a in Table 2), 30°C, 230rpm, react for 2h. Take 1ml of the reaction solution, add 800μl of ethyl acetate to extract, centrifuge at 12,000 for 2min, take the organic phase, add appropriate amount of anhydrous sodium sulfate to dry, distill under reduced pressure, dissolve the product in 1ml of isopropanol, and filter by organic membrane for HPLC detection. The ShimadzuProminenceLC-20ADsystem Daicel chiral chromatography column AD-H is used, and the detector is PDA. Detection conditions, isopropanol:n-hexane=10:90, 0.5ml / min, column temperature 35°C. The product is (3R,4S)-4-phenyl-3,4-epoxy-2-butanone, the conversion rate is >99%, and the enantioselectivity ee value is >99%. The product was dete...

Embodiment 3

[0046] Embodiment 3: Synthesis of other chiral allyl epoxy ketones

[0047] Take 2 g of the bacteria from Example 1 and resuspend in 10 ml potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer (0.1 M, pH=7.0), add 10 mM substrate, react at 30° C., 230 rpm for 2 h. The reaction solution treatment method is the same as in Example 2.

[0048] Substrates include 4-phenyl-3-buten-2-one and derivatives obtained by halogen and methyl substitution at o- / m- / p-position respectively (Table 2). Conversion 70-99% and ee 85-99%. It has good conversion efficiency and excellent enantioselectivity ee value (>99%) for most substrates.

[0049]

[0050] Table 2

[0051]

[0052]

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Abstract

The invention discloses a new method for using controllable multi-enzyme cascade reaction to synthesize two kinds of optical pure epoxy compounds. The method is characterized in that coexpression carbonyl reductase in escherichia coli and the recombinant bacteria of styrene monooxygenase are used as biocatalysts, alpha, beta-unsaturated ketone compounds are used as substrate, isopropanol is used as a switch for controlling, and chirality pure allylic epoxy ketone / alcohol is synthesized.

Description

[0001] manual technical field [0002] The invention belongs to the technical field of microorganism and enzyme engineering, and specifically relates to a recombinant Escherichia coli that co-expresses carbonyl reductase and styrene monooxygenase as a biocatalyst to catalyze the synthesis of optically pure allylic rings from ɑ, β-unsaturated ketones. Oxyketone or alcohol approach. Background technique [0003] Optically pure epoxy compounds are one of the most important building blocks in organic synthesis, and numerous chiral functional groups can be derived by ring opening with nucleophiles. Optically pure allyl epoxy ketone / alcohol is a special multifunctional epoxy compound with carbonyl / hydroxyl in the ortho position. It not only has the common properties of epoxy compounds, but also has the special reactivity of hydroxyl / carbonyl. Therefore, they have a wider range of derivatization. A large number of chiral functional groups can be derived through functional group tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/02C12N1/21C12N15/70C12R1/19
Inventor 吴中柳刘育昌
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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