Biological preparation method of intermediate of atazanavir

A technology for biological preparation and intermediates, applied in fermentation and other directions, can solve problems such as violation of basic principles, decreased enzyme activity and stability, and unfavorable energy conservation and environmental protection.

Inactive Publication Date: 2013-12-25
ENZYMEWORKS
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  • Abstract
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Problems solved by technology

Because this method will use volatile isopropyl alcohol, produce volatile acetone by-product simultaneously, need carry out pressure control at higher temperature, process stability control is more difficult; Higher temperature has violated the basic principle of green chemistry simultaneou

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  • Biological preparation method of intermediate of atazanavir
  • Biological preparation method of intermediate of atazanavir
  • Biological preparation method of intermediate of atazanavir

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[0023] The invention provides an improved biological preparation method of atazanavir intermediates, which solves the danger and pollution problems in the production of atazanavir intermediates by chemical methods. Compared with chemical methods, its advantage is that it does not use flammable and Explosive chemical reagents do not use a large amount of organic solvents, which reduces pollution; the method of the invention also solves the problems of low substrate concentration, high energy consumption, and low efficiency in the production of atazanavir intermediates by traditional biological methods. Almac's technology has obvious advantages in substrate concentration. Compared with Codexis technology, the substrate concentration and enzyme dosage are at the same level, but by improving the coenzyme cycle system, the reaction temperature is lowered, the control risk is reduced, and a green and energy-saving reaction is realized.

[0024] The reaction flow chart of the inventi...

Embodiment 1

[0028] Embodiment 1 (enzyme reaction small test)

[0029] Add 100 mg of substrate ((S)-tert-butyl(4-chloro-3-carbonyl-1-phenylbutyl-2-yl)carbamate), 400 μL of co-solvent acetonitrile, 100 mg of glucose into a 5 mL reactor Stir well; weigh 3 mg of ketoreductase (KRED) enzyme powder, 3 mg of glucose dehydrogenase (GDH) and 0.36 mg of NAD in 1400 μL of pH 8.5 triethanolamine buffer solution, and add this mixed solution into the reactor Stir and start the reaction in a water bath at 33°C; take a sample for HPLC detection after 1 hour, and the conversion rate is 37%, and take a sample for HPLC detection after 20 hours, and the conversion rate is 96% (see figure 1 and figure 2 , the product corresponding to 7.48 minutes in the figure, and the substrate corresponding to 8.08 minutes).

Embodiment 2

[0030] Example 2 (enzyme reaction amplification)

[0031] Add 100g substrate ((S)-tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate), 170mL toluene and 100g glucose in 2L reactor, stir Uniform; Weigh 1.2g ketoreductase (KRED) enzyme powder, 1.2g glucose dehydrogenase (GDH) enzyme powder and 0.15g NAD and dissolve in 585mL pH8.5 triethanolamine buffer solution, and add this mixed solution to Stir in the reactor and start the reaction in a water bath at 33°C; after 24 hours, the conversion rate of the substrate is 97% as detected by HPLC. Add ethyl acetate and continue to stir and extract, combine the organic phases, dry and filter, and concentrate to obtain 85g of solid product, with a yield of 85%, a purity of 97.7%, and an optical purity of 99.8% (chiral HPLC) (chiral HPLC racemate standard product such as image 3 As shown, the product is as Figure 4 shown).

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Abstract

The invention relates to a biological preparation method of an intermediate of atazanavir. (S)-tertiary butyl (4-chloro-3-carbonyl-1-benzene butyl-2-yl) carbamic acid ester is taken as a substrate. The preparation method comprises the following steps of adding the substrate, a cosolvent and glucose into a reactor, stirring uniformly, weighing ketoreductase powder, glucose dehydrogenase and cofactor, dissolving in a water phase buffered solution with pH of 8-9, adding the obtained mixed solution into the reactor, stirring, controlling the temperature to be at 32DEG C-34 DEG C, and beginning reacting, wherein at the beginning of the reaction, the mass volume ratio concentration of the substrate is 0.05-0.2g/ml, and the mass ratio of the added ketoreductase to cofactor to glucose to the substrate is (0.02-0.03):(0.001-0.005):(0.8-1.2):1. Compared with the prior art, the biological preparation method realizes more environment-friendly and mild biological conversion process with high reaction efficiency, and has important application value.

Description

technical field [0001] The invention belongs to the technical fields of biopharmaceuticals and biochemical engineering, and in particular relates to a biological preparation method of an atazanavir intermediate. Background technique [0002] Atazanavir (Atazanavir, CAS: 198904-31-3) is a protease inhibitor antiviral drug researched and developed by Bristol-Myers Squibb. Its trade name is Reyataz, and it is usually used together with other drugs. for the treatment of AIDS. After the FDA approved the listing in 2003, the global sales of the drug in 2009 were 5.2 billion US dollars, ranking 70th. With the frequent outbreaks of "SARS", "A" and bird flu worldwide and the deepening of AIDS prevention and control work, people's demand for antiviral drugs continues to increase. It is foreseeable that the sales and ranking of such drugs will further rise. [0003] The molecular structure of the main active ingredient of atazanavir is relatively complex, but the two chiral centers ...

Claims

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Application Information

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IPC IPC(8): C12P13/02
Inventor 尹将来鞠鑫李斌陶军华
Owner ENZYMEWORKS
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