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A kind of efficient preparation method of heterocyclic drug intermediate

A technology of heterocycles and intermediates, applied in the field of biocatalysis, can solve the problems of increasing the production cost of catalysts, adding more enzymes and organic solvents, unfavorable amplification and application, etc., and achieves the effect of reducing the product inhibition effect.

Active Publication Date: 2021-08-31
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0013] (11) Patent CN201310173088.2 discloses a method of using recombinant ketoreductase (KRED) enzyme powder to asymmetrically reduce N-B OC -3-piperidone, but did not disclose the gene sequence or amino acid sequence of ketone reductase (KRED)
Patent CN201310054684.9 discloses an asymmetric synthesis of (S)-1-tert-butoxycarbonyl-3-hydroxypiperidine using alcohol dehydrogenase PAR, but the organic reagent isopropanol is used for the coenzyme cycle, and the organic reagent There is a greater degree of damage to the enzyme activity, and it has obvious inhibitory effect
Patent CN201610132936.9 discloses a method using carbonyl reductase R E The CR enzyme asymmetrically reduces ketoreductase (KRED), but this enzyme requires N I -NTA purification, and it uses 2-octanol-water two-phase reaction, which is not conducive to large-scale production or relatively high production cost
Pichia pastoris P reported in patent CN108220358A ICHIA SP.SIT2014 can be used as a biocatalyst for preparing (S)-NBHP, but too much catalyst addition increases the production cost
CN10822061A reports that ketoreductase MT-KRED is used for preparing (S)-NBHP, but needs to add expensive coenzyme in reaction process
[0014] Although the ketoreductase reported above can be used to prepare (S)-NBHP, the reaction process requires the addition of expensive coenzyme, a large amount of enzyme and organic solvents, which is not conducive to the scale-up application in the actual industry.

Method used

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  • A kind of efficient preparation method of heterocyclic drug intermediate
  • A kind of efficient preparation method of heterocyclic drug intermediate
  • A kind of efficient preparation method of heterocyclic drug intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Rational mutation and mutant construction of key amino acids of alcohol dehydrogenase KpADH include the following steps:

[0044] 1. Identify key amino acids that control stereoselectivity and catalytic activity

[0045] Through the crystal structure (PDB: 5Z2X) derived from Kluyveromyces alcohol dehydrogenase KpADH, find out the amino acid that interacts with the substrate NBPO; through the conservative analysis of amino acid sequence alignment with more than 30% homology, screen out Key amino acid residue 127.

[0046] 2. Construction of Mutants

[0047] Using the pET28a-KpADH recombinant plasmid preserved in the laboratory as a template (recorded in the patent application with publication number CN105936909A), the 127th amino acid of the alcohol dehydrogenase KpADH with amino acid sequence such as SEQ ID No.1 was carried out by using the whole plasmid PCR method Site-directed saturation mutagenesis. The mutants Y127A, Y127C, Y127F, Y127I, Y127M, Y127Q, Y127V, Y127...

Embodiment 2

[0049] According to Example 1, the mutants with improved catalytic activity were induced, expressed, purified and kinetically determined, including the following steps:

[0050] 1. Induced expression

[0051] The mutant in Example 1 was inoculated into 50 μL / mL LB medium, and cultured with shaking at 37° C. and 180 rpm. When OD600 reached 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG, 0.2M) was added to a final concentration of 0.2mM, and the temperature was lowered to 25°C to induce protein expression. At the end of the culture, cells were harvested by centrifugation and sonicated in PBS buffer (pH 7.4). The cell lysate was centrifuged at 8000rpm for 30min.

[0052] 2. Protein purification

[0053] Nickel column affinity chromatography is used. According to the His-Tag tag at the N-terminal of KpADH, it can competitively bind with nickel, and gradient or linear elution methods can be used. KpADH and its mutants could be completely eluted when the imidazole concentration ...

Embodiment 3

[0061] In order to explore the heterocyclic ketone substrate spectrum of KpADH and its mutants, the heterocyclic ketones dihydro-3(2H)-furanone, tetrahydrothiophen-3-one, cyclohexanone, 4-ethylcyclohexanone, N -Boc-3-pyrrolidone, N-Boc-2-piperidone, N-Boc-3-piperidone, N-Boc-4-piperidone were used to determine the substrate kinetics of KpADH and mutants to heterocyclic ketones study. As shown in Table 2, except for the N-Boc-2-piperidone substrate in the ortho position, it exhibits catalytic activity for other substrates. Mutant Y127W in the substrate spectrum, the k of the substrate N-Boc-3-pyrrolidone cat / K m from 0.2s -1 mM -1 increased to 2.1s -1 mM -1 , and the k for substrate 7a cat / K m from 3.6s -1 mM -1 increased to 31.0s -1 mM -1. At the same time, the e.e. values ​​for the two substrates were also increased to over 99%. This indicated that the mutant Y127W specifically increased the catalytic efficiency of substrates with a Boc group and a carbonyl gr...

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Abstract

The invention discloses a high-efficiency preparation method of a heterocyclic drug intermediate. The present invention couples alcohol dehydrogenase mutants and glucose dehydrogenases to catalyze heterocyclic substrates to generate heterocyclic drug intermediates. The alcohol dehydrogenase mutants have an amino acid sequence as shown in SEQ ID NO.2 Tyrosine at position 127 of the parent alcohol dehydrogenase shown is mutated to tryptophan. The mutant Y127W of the present invention can alleviate the product inhibition effect in a single aqueous phase system without adding any co-solvent, so that the conversion rate can reach more than 99% within 12 hours. Coupling the mutant Y127W with glucose dehydrogenase (BmGDH) achieved a 50 mL scale in a single aqueous phase system without adding any exogenous coenzymes and organic co-solvents, and the substrate concentration was as high as 600 g L ‑1 Gram-level preparation of , the catalyst loading was 3.3%. The e.e. value of the final product (S)-NBHP is as high as 99.4%, and the space-time yield is about 1400g L ‑1 d ‑1 , the product purity is 99.58%.

Description

technical field [0001] The invention relates to a high-efficiency preparation method of a heterocyclic drug intermediate, belonging to the technical field of biocatalysis. Background technique [0002] (S)-N-B OC -3-Hydroxypiperidine [(S)-NBHP] is a key chiral intermediate in the synthesis of the drug Ibrutinib (IBRUTINIB) for the treatment of lymphoma. The method of biosynthesis of (S)-NBHP is more green and environment-friendly, thus attracting more and more attention. However, previous studies have shown that the biosynthesis of (S)-NBHP requires the addition of organic co-solvents and expensive coenzymes, and the high concentration of substrates will lead to substrate or product inhibition, which will increase the cost of (S)-NBHP synthesis. [0003] (1) 2009A CHERETZ et al., used the method of biocatalytic synthesis for the first time, using the reductase in carrot cubes for catalysis. The catalyst was cheap and environmentally friendly, and provided a new idea for t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12P17/12
CPCC12N9/0006C12P17/12C12Y101/01001
Inventor 倪晔吴彦霏周婕妤许国超韩瑞枝
Owner JIANGNAN UNIV
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