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Synthesis method of saxagliptin chiral intermediate

A sequence and carrier technology, applied in the field of biopharmaceuticals, can solve problems such as difficult to increase production capacity, decreased thermal stability, and high energy consumption

Active Publication Date: 2014-02-05
弈柯莱(台州)药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The phenylalanine dehydrogenase (PDH) used in the reductive ammoniation process is modified from the N-terminus and the C-terminus, and its enzyme activity on ketoacid substrates is increased, but its thermal stability is decreased. The reaction time at high temperature is too long, it takes more than 38 hours to achieve complete conversion, the energy consumption is too high, and the production capacity is difficult to increase

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  • Synthesis method of saxagliptin chiral intermediate
  • Synthesis method of saxagliptin chiral intermediate
  • Synthesis method of saxagliptin chiral intermediate

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Experimental program
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Effect test

Embodiment 1

[0037] The enzyme activity assay of embodiment 1PDH and FDH

[0038] The enzyme activity of PDH was measured at 30°C, and the reaction system contained 0.4mM NADH, 50mM keto acid A1 (dissolved in 1 equivalent of NaOH), and 0.75M ammonia water (adjusted to pH 8.75 with HCl). After adding phenylalanine dehydrogenase, the change of the ultraviolet absorption value of the reaction system was detected at 340 nm.

[0039] The enzyme activity of FDH was measured at 30°C. The reaction system contained 1mM NAD, 100mM ammonium formate, and 100mM potassium phosphate (pH8.0) buffer solution. After adding formate dehydrogenase, the change of UV absorption value at 340nm was detected within 30 minutes. .

[0040] Enzyme activity definition:

[0041] Every 0.1 change in the UV absorbance value at 340nm corresponds to a 0.016mM change in the concentration of NADH. Therefore, the enzyme activity calculation formula is obtained:

[0042] U = A ...

Embodiment 2

[0046] Embodiment 2 substrate and product analysis method

[0047] The product was detected by high-performance liquid chromatography (HPLC): water and acetonitrile (45:55) were used as the mobile phase, the chromatographic column was ODS-18 reversed-phase column, Shimadzu LC-15C high-performance liquid chromatography, and ultraviolet absorption was detected at 210nm ; The reaction system was diluted with water and acetonitrile (45:55), centrifuged and filtered with a nylon membrane, then injected for detection, the retention time of the keto acid substrate 2-(3-hydroxy-1-adamantyl)-2-glyoxylic acid The peak retention time of the amino acid product was 4.19 minutes, and the retention time of the Boc-protected amino acid was 2.1 minutes.

[0048] Optical purity e.e. values ​​were analyzed on Agilent 1260 series HPLC using AD-H chiral column (Diacel Chemical). The retention time of the R-configuration product was 3.5 min, and the retention time of the S-configuration product was...

Embodiment 3

[0050] The cloning of embodiment 3PDH and construction E.coli mutant library

[0051] The pdh gene of Geobacillus sp. Y412MC61 was introduced into E.coli. To amplify the pdh gene (SEQ ID No.2) encoding the PDH enzyme (SEQ ID No.1), use primers F1 (SEQ ID No.3) and R1 (SEQ ID No.4) as forward and reverse, respectively primers. Both primers contained sites compatible with the PCR-amplified deoC gene fragment obtained by site-directed recombination cloning using Gateway Technology. The gel electrophoresis patterns of the PCR amplification products are as follows: figure 1 shown. Using a random mutagenesis kit, by changing the MnSO 4 concentration to carry out multiple reactions, thereby introducing 1 to 3 point mutations into the pdh gene (SEQ ID No.2) of Geobacillus sp.Y412MC61, so that 1-3 amino acid residues in the amino acid sequence of the PDH enzyme are replaced.

[0052] Sequence of forward primer F1 (SEQ ID No.3):

[0053] 5'-GAGCATATGAATGTCATGCTATCGCC-3'

[0054] ...

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Abstract

The invention provides a phenylalanine dehydrogenase (PDH) mutant derived from geobacillus, which is high in enzyme activity and thermal stability compared to wild PDH. Furthermore, the invention provides a method for catalytic synthesis of a saxagliptin chiral intermediate, namely (S)-N-t-butyloxycarbonyl-3-hydroxy-1-adamantyl-D-glycine (Boc-HAG) through the PDH mutant. According to the method provided by the invention, Boc-HAG can be directly prepared through a reaction in two steps, and e.e. (enantiomeric excess) value exceeds 99.9%; generation of side products can be reduced, yield of 95% can be achieved within 12 hours, catalysis time is greatly shortened, energy consumption is reduced and a post-treatment process is simplified.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a method for synthesizing a key chiral intermediate of saxagliptin. Background technique [0002] Saxagliptin is a highly efficient, selective and competitive dipeptidyl peptidase-IV (DPP-IV) inhibitor jointly developed by Bristol-Myers Squibb and AstraZeneca. It was approved by the European Medicines Agency in 2009 ( EMA) and the U.S. Food and Drug Administration (FDA) approved for marketing, the product name is Onglyza, and its chemical structure is as follows: [0003] [0004] Saxagliptin (Onglyza) is the first DPP-IV inhibitor approved for marketing in the European market. It has submitted new drug registrations in 90 countries around the world and has been approved in 56 countries, including the United States, Canada, and Chile. , India, Brazil and 30 countries of the European Union. Due to its small side effects and easy dosage form, it has grown rapidly since its la...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12P15/00C07D209/52C12R1/01
CPCC12N9/0018C12P15/00C12P17/10C12Y104/0102
Inventor 罗煜丁时诚瞿旭东
Owner 弈柯莱(台州)药业有限公司
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