Pseudomonas pseudoalcaligenes and application of pseudomonas pseudoalcaligenes to preparation of sitagliptin intermediate

Abstract

The invention discloses pseudomonas pseudoalcaligenes and application of the pseudomonas pseudoalcaligenes to preparation of a sitagliptin intermediate. A colony of the pseudomonas pseudoalcaligenes XW-40 is flat and round in surface morphology, smooth and neat in edge, moist, milk white and opaque and is about 1.9mm in diameter. The pseudomonas pseudoalcaligenes is capable of effectively catalyzing 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5)-trifluorophenyl)-butan-2-one to be reduced into a sitagliptin chiral intermediate; in optimal reaction conditions, the pseudomonas pseudoalcaligenes can be tolerant to 10 g / L of high substrate concentration while keeps high selectivity, and is weak in substrate inhibition effect, so that high-substrate-concentration biological catalysis can be achieved; the pseudomonas pseudoalcaligenes is recyclable and removes inhibition effect of a substrate and a product on cells as compared with fed-batch, so that catalytic activity of the pseudomonas pseudoalcaligenes is used maximally.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to an alcaligenes-like pseudomonas strain and its application in preparing sitagliptin intermediates. Background technique [0002] Sitagliptin phosphate is the first dipeptidyl peptidase-4 (DPP-4) inhibitor developed by Merck for the treatment of type 2 diabetes, which can effectively target insulin in type 2 diabetes Resistance and dysfunction of islet α and β cells can slow down the degradation of incretin GLP-1 by inhibiting DPP-4, thereby exerting a hypoglycemic effect. Compared with traditional oral hypoglycemic drugs that easily cause side effects such as hypoglycemia, weight gain, and nausea and vomiting, this inhibitor has obvious advantages and a good market prospect. There are two key chiral intermediates of sitagliptin phosphate, one is a chiral β-amino acid, and the other is its corresponding chiral alcohol intermediate. The current focus of the ...

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Problems solved by technology

[0007] (4) Use isopropylamine as ammonia donor and sitagliptin precursor ketone as ammonia acceptor to prepare sitagliptin by transaminase biocatalysis. This method has the advantages of environmental friendliness and mild reaction conditions, but the special enzyme is not easy The high nature of acquisition and price (separation schemes, etc.) cannot be ignored;

[0008] (5) R-beta-aminobutyric acid is obtained by condensation, reduction, biological enzyme resolution, hydrolysis, protection of amino groups, diazotization, Arndt-Fistert rearrangement reaction, and hydrolysis with trifluorobenzaldehyde and N-acetylglycine, but The synthetic route is too long, the yield of chiral amino acid obtained by chiral resolution is not more than 50%, and the recycling of the other half of the enantiomers is a big problem, and the reaction process uses diazomethane, diazomethane Unstable toxic gas at room temperature, explosive, dangerous to handle

[0010] (1) WO09 / 045507 uses an appropriate carbonyl reductase to asymmetrically reduce the β-carbonyl part to obtain a β-hydroxyl intermediate, which is then reacted with an azetidinone intermediate to obtain sitagliptin. The disadvantages of this method are: Reaction at high pressure, use of very expensive metal chiral catalysts (Rh or Ru), low stereoselectivity and rhodium-contaminated products and resulting final compounds are difficult to purify;

[0011] (2) WO12 / 046254 discloses a method for preparing sitagliptin intermediates through enzymatic conversion, and provides two methods for biocatalyzed synthesis of chiral alcohol intermediates, which are produced by engineering bacteria whole cells and engineering bacteria, respectively. The crude extract of carbonyl reductase is used as a biocatalyst to reduce sitagliptin precursor ketone to the corresponding (S) configuration alcohol; the method has the advantages of environmental friendliness, mild reaction conditions, and high ee value, but in other On the one hand, the construction of engineering bacteria is very difficult and expensive. The problem of using crude enzymes as biocatalysts is that the free crude enzymes are unstable in the reaction solution, the catalytic ability of the enzymes is low, and the enzymes are easily inactivated. It is not easy to realize the recycling of enzymes, and the separation operation in the later stage is complicated; while using the whole cell as a catalyst, the problem of dissolving the substrate in toluene for two-phase catalytic reaction is that the solubility of the substrate in toluene is small, and the conversion of the reaction The substrate concentration is low, not suitable for large-scale industrial production

[0012] Existing chemical method and biocatalytic method synthetic sitagliptin and the method for intermediate all have its weak point, therefore, the method for the intermediate of preparation sitagliptin of environmental friendliness of development is very significant

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