Continuous flow method for preparing (r)-3-hydroxy-5-hexenoate

Patent metadata

Field	Value
Publication number	US-2022033863-A1
Application number	US-202117504858-A
Country	US
Kind code	A1
Filing date	Oct 19, 2021
Priority date	Oct 22, 2020
Publication date	Feb 3, 2022
Grant date	—

Abstract

Disclosed herein relates to biopharmaceuticals, and more particularly to a continuous flow method for preparing (R)-3-hydroxy-5-hexenoate. Carbonyl reductase and isopropanol dehydrogenase are co-immobilized onto an inert solid medium simultaneously to prepare a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, which is then filled into a microchannel reactor of the micro reaction system. A solution containing substrate 3-carbonyl-5-hexenoate is subsequently pumped into the microchannel reactor to perform an asymmetric carbonyl reduction reaction to obtain (R)-3-hydroxy-5-hexenoate.

First claim

What is claimed is:

1 . A continuous-flow method for preparing (R)-3-hydroxy-5-hexenoate using a micro reaction system, the micro reaction system comprising a microchannel reactor, wherein the method comprising:
(1) Co-immobilizing a carbonyl reductase and an isopropanol dehydrogenase onto an inert solid medium simultaneously to prepare a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst; and filling the microchannel reactor with the co-immobilized catalyst; (2) pumping a substrate solution containing a 3-carbonyl-5-hexenoate into the microchannel reactor filled with the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst prepared in step (1) to perform an asymmetric carbonyl reduction reaction; and (3) collecting the reaction mixture flowing out of the microchannel reactor followed by separation and purification to obtain a target product (R)-3-hydroxy-5-hexenoate; wherein the (R)-3-hydroxy-5-hexenoate is shown in formula (I), and the 3-carbonyl-5-hexenoate is shown in formula (II); and the asymmetric carbonyl reduction reaction is shown in the following reaction scheme:

wherein R is linear or branched C1-C8 alkyl group, linear or branched C3-C8 cycloalkyl group, monosubstituted or polysubstituted aryl group, or monosubstituted or polysubstituted aralkyl group.

2 . The method of claim 1 , wherein in step (1), the inert solid medium is a composite material of polyvinyl alcohol and polyethylene glycol; and the step of “co-immobilizing a carbonyl reductase and an isopropanol dehydrogenase onto an inert solid medium simultaneously to prepare the co-immobilized catalyst” comprises:
(a) preparing an aqueous solution of the polyvinyl alcohol and the polyethylene glycol; heating the aqueous solution until the aqueous solution becomes clear; and cooling the aqueous solution to 50° C. or less to obtain a first solution;
(b) adding a crude carbonyl reductase solution and a crude isopropanol dehydrogenase solution into the first solution followed by uniform mixing to obtain a second solution; and
(c) dropwise adding the second solution onto a polyethylene film; drying the polyethylene film at 35-40° C. for 0.5-1 hour to obtain the co-immobilized catalyst; and storing the co-immobilized catalyst at 4° C. for later use;
wherein an amino acid sequence of the carbonyl reductase is shown in SEQ ID NO: 1; and an amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID NO: 2;
a weight ratio of the polyvinyl alcohol to the polyethylene glycol is 5:1-3;
the crude carbonyl reductase solution and the crude isopropanol dehydrogenase solution both have an initial concentration of 10%-30% (w/v); and
in step (b), a volume ratio of the crude carbonyl reductase solution to the crude isopropanol dehydrogenase solution to the first solution in the second solution is 2:1:5-10.

3 . The method of claim 1 , wherein the microchannel reactor is a tubular microchannel reactor or a plate-type microchannel reactor.

4 . The method of claim 3 , wherein the microchannel reactor is a tubular microchannel reactor with an inner diameter of 100 μm-20 mm; and
the microchannel reactor is a plate-type microchannel reactor having a reaction fluid channel with a hydraulic diameter of 100 μm-20 mm.

5 . The method of claim 1 , wherein in step (2), the substrate solution is prepared by dissolving 3-carbonyl-5-hexenoate (II), isopropanol, coenzyme NADP + and an organic solvent into a phosphate buffered solution (PBS); and
the organic solvent is a polar aprotic solvent; the organic solvent is N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, sulfolane, 1,3-dimethyl-2-imidazolinone, hexamethylphosphoric triamide, acetonitrile and a ketone solvent.

6 . The method of claim 5 , wherein the substrate solution comprises 1-100 g/L of 3-carbonyl-5-hexenoate (II), 1-50 g/L of isopropanol, 0.005-0.1 g/L of the coenzyme NADP + and 20-250 g/L of the organic solvent.

7 . The method of claim 5 , wherein the phosphate buffered solution is an aqueous solution of a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate or potassium dihydrogen phosphate; and
the phosphate buffered solution has a pH of 6-8.

8 . The method of claim 1 , wherein in step (2), the asymmetric carbonyl reduction reaction is performed at 15-40° C.; and a residence time of the substrate solution in the microchannel reactor is 0.1-30 min.

9 . The method of claim 1 , wherein the micro reaction system further comprises a feeding pump and a back pressure regulator; an inlet of the microchannel reactor is connected to the feeding pump, and an outlet of the microchannel reactor is connected to the back pressure regulator; and a back pressure range of the back pressure regulator is 0.1-3 MPa.

10 . The method of claim 9 , wherein the microchannel reactor is further sequentially connected to a microfluidic liquid-liquid extractor and a liquid-liquid membrane separator to achieve continuous enzymatic reaction, liquid-liquid extraction and separation.

11 . The method of claim 9 , wherein the microchannel reactor is further connected to a multi-stage extraction and separation unit to achieve continuous enzymatic reaction, liquid-liquid extraction and separation. The multi-stage extraction and separation unit is composed of at least two pairs of microfluidic liquid-liquid extractor and liquid-liquid membrane separator that are sequentially connected in series.

12 . The method of claim 9 , wherein the microchannel reactor is further connected to a centrifugal extractor or at least two centrifugal extractors installed in series to achieve continuous enzymatic reaction, liquid-liquid extraction and separation.

13 . A micro reaction system for preparing (R)-3-hydroxy-5-hexenoate, comprising:
a feeding pump; a microchannel reactor; and a back pressure regulator; wherein an inlet of the microchannel reactor is connected to the feeding pump, and an outlet of the microchannel reactor is connected to the back pressure regulator; the microchannel reactor is filled with a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst, and the carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst is prepared by co-immobilizing a carbonyl reductase and an isopropanol dehydrogenase onto an inert solid medium simultaneously; the feeding pump is configured to pump a substrate solution containing 3-carbonyl-5-hexenoate into the microchannel reactor to perform an asymmetric carbonyl reduction reaction; and the back pressure regulator is configured to provide a pressure to the microchannel reactor; and the reaction mixture is discharged from the back pressure regulator to afford a target product (R)-3-hydroxy-5-hexenoate; wherein the (R)-3-hydroxy-5-hexenoate is shown in formula (I):

the 3-carbonyl-5-hexenoate is shown in formula (II):

wherein R is linear or branched C1-C8 alkyl group, linear or branched C3-C8 cycloalkyl group, monosubstituted or polysubstituted aryl group, or monosubstituted or polysubstituted aralkyl group; and
the microchannel reactor is a tubular microchannel reactor or a plate-type microchannel reactor.

Assignees

• Univ Fudan

Inventors

• Chen Fener
• Cheng Dang
• Huang Zedu
• Hu Chen
• Jiang Meifen
• Liu Minjie
• Huang Huashan

Classifications

• Y02P20/50

  Improvements relating to the production of bulk chemicals · CPC title

• C12P7/62 Primary

  Carboxylic acid esters · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12P41/002 Primary

  by oxidation/reduction reactions · CPC title

• C12N11/084

  Polymers containing vinyl alcohol units · CPC title