Method for catalyzing to synthesize Atazanir intermediate and application thereof
A technology of atazanavir and intermediates, which is applied in the field of catalytic synthesis of atazanavir intermediates, can solve the problems of low reaction efficiency, unreusable carbonyl reductase, and high requirements for reaction conditions, and achieves high cumulative product concentration and convenient Separation and purification, the effect of reducing energy consumption
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Embodiment 1
[0036] This example discloses a method for catalytically synthesizing an atazanavir intermediate, using (S)-tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate as Substrate, with carbonyl reductase NaSDR and coenzyme as catalyst, catalyzed synthesis of tert-butyl ((2S,3R)-4-chloro-3-hydroxyl-1-benzene in the system where co-substrate, cosolvent and buffer exist Butyl-2-yl) carbamate; the carbonyl reductase is immobilized on the resin.
[0037] Specifically include the following steps:
[0038] S1: immobilizing carbonyl reductase on the resin;
[0039] S2: Substrate (S)-tert-butyl (4-chloro-3-carbonyl-1-phenylbutyl-2-yl) carbamate, co-substrate, co-immobilized enzyme system, co-solvent and buffer solution into the reactor, the reaction pH value is 8.0-11.0, and the reaction temperature is 25-30°C. After 3-6 hours of reaction, tert-butyl ((2S,3R)-4-chloro-3-hydroxy-1 -phenylbutyl-2-yl)carbamate.
[0040] Step S1 includes:
[0041] S11: Soak the resin in TEA buffer...
Embodiment 2
[0049] This embodiment discloses a method for preparing a crude enzyme solution of carbonyl reductase NaSDR, which specifically includes the following steps:
[0050] 1. Activation of E.coli-NaSDR bacteria
[0051] On the LB solid plate containing kanamycin (50μg / ml), the bacterial solution was coated by the method of partitioning or dilution. After culturing at 37°C for 20 hours, a single colony of activated E.coli-NaSDR was obtained.
[0052] 2. Fermentation and cultivation of E.coli-NaSDR bacteria
[0053] (1) Inoculate a single colony of E.coli-NaSDR bacteria into 5ml primary seed medium, and cultivate it at 37°C and 220rpm for 11 hours, and the OD600 reaches 0.9-1.2, which is the primary seed solution.
[0054](2) Inoculate 5 ml of the primary seed solution into the fermentation medium (2.5% inoculum size), and cultivate at 37° C. and 220 rpm for 2-3 hours. When the OD600 of the fermentation medium reaches 0.9-1.2, add 400 μL of IPTG (isopropylthiogalactopyranoside, co...
Embodiment 3
[0058] This embodiment discloses a resin pretreatment method, the specific steps are as follows:
[0059] Soak 1 g of resin in pH 10.5 TEA buffer for 1 hour, remove supernatant and floating impurities, add 500ul of 25% glutaraldehyde to the resin, add TEA buffer to a final volume of 10ml, make the volume of glutaraldehyde The score is 1.25%. Under the condition of 30°C, place it on a shaker at 230 rpm for 4 hours, then filter with filter paper to obtain the activated resin, rinse the activated resin three times with TEA buffer to remove glutaraldehyde, and drain it for later use.
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Abstract
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