(4S)- 3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one : Biosynthesis, Optimization of biosynthesis conditions And Influencing factors Studies

General description

The (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one, with the CAS No:189028-95-3, is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe, which is the first selective cholesterol absorption inhibitor that reduces plasma LDL cholesterol levels and increases plasma HDL levels. Therefore, synthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one has attracted more and more attention. High-yield chemical synthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one has been extensively studied. This chemical molecular formula is C20H20FNO4 and molecular weight is 357.38.[1]

Figure 1 the molecular formula of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one

Preparation

The construction of the chiral amino group of the intermediate, (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one from (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone was considered as the key step to produce ezetimibe. The traditional chemical synthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one requires expensive noble metal catalyst and harsh reaction conditions (ultra-low temperature, -80 to-75℃), which results in low overall yield, high production cost, poor security, and contaminant discharge.[2-3] To explore for the synthesis approaches which meet the concepts of green chemistry is of great value.[4] Efficient enzymatic methods were developed for the synthesis of (S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one, by transesterification of (RS)-3-[5-(4-fluorophenyl)-5-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one[(R,S)-F0P alcohol] and hydrolysis of (RS)-l-(4-fluorophenyl)-5-oxo-5-[(S)-2-oxo-4-phenyloxazolidin-3-yl]pentyl acetate[(R,S)-FOP acetate] using lipase as enzyme source. The synthesized S-diastereomer is an intermediate for the potent cholesterol absorption inhibitor, ezetimibe. Among various lipases tried, Candida rugosa lipase in diisopropyl ether was best for both the reactions. Vinyl acetate was found as suitable acyl donor in transesterification reaction. A higher amount of enzyme (500mg) was required for the transesterification of 10mM substrate; it may be due to the enzyme denaturation by acetaldehyde formed in the reaction. The este r hydrolysis reaction worked well, excellent conversion and were obtained at 40°C, pH7. The 300 mg enzyme hydrolyzed 120 mg (R,S)-FOP acetate with 50% conversion.[3]

Recently, biocatalyst has attracted more and more attention due to its wide range of natural sources, mild reaction conditions, high catalytic efficiency, excellent stereoselectivity, and environmental friendship.[5] Carbonyl reductases (CRs, EC) are a type of nicotinamide coenzyme-dependent oxidoreductases which catalyze the reduction of a carbonyl group to the corresponding chiral alcohol, in the present of the reduced form of the cofactor, NAD[P]H.[3,6-7] CRs have been employed as a valuable biocatalyst for ezetimibe synthesis. As early as 1997, it was reported that (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one was synthesized by whole cells of Schizosaccharomyces octosporus (ATCC 2479) and S. octosporus (ATCC 4206) containing CRs with the yields of 33.0 and 34.0%, respectively, and the e.e. of 99.0%, under the substrate ET-4 concentration of 1 gL-1[8] Subsequenly, Homann et al. Employed Zygosaccharomyces baili (ATCC 38924) cells containing CR to catalyze the completely biotransformation of 10g/L ET-4 to (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one with the e.e. of 95.0%.[9] In previous study, a recombinant carbonyl reductase CR125 was obtained from Lactobacillus kefiri to catalyze 150g/L ET-4 to (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one with the conversion of 99.1% and the e.e. 99.9%.[10] In another study, in order to enhance the NADPH regeneration and to further improve the catalytic efficiency, a recombinant plasmid containing CR (CR125) and GDH from Exiguobacterium sibiricum was constructed and coexpressed in E. coli BL21 (DE3) cells to achieve the simultaneous ET-4 asymmetric reduction and in situ NADPH regeneration. To our knowledge, this is the first report about the coexpression of CRs and GDH for the efficient biosynthesis of ezetimibe intermediate.[11]

Optimization of biosynthesis conditions for (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one:Certain p-xylene solution reagent containing 100g/L ET-4 and equal volume of 100mM Na2HPO4-NaH2PO4 buffer containing glucose with the mole ratio of 1.2:1 to the substrate were mixed.20 Recombinant cells containing CR125 and GDH (20g DCW/L) were replenished into a 100ml reaction system to initiate the reaction at 35℃ 600 rpm, pH 7.0, and the incubation continued until the reaction was completed. The pH was adjusted to 7.0 with 2 M NaOH or HCI during the reaction using a 902 Titrando (Metrohm, Switzerland). Optimization of biosynthesis conditions for (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one was based on the cosolvent of dimethyl sulfoxide(DMSO) concentration (0-25%), glucose concentration (1.0-2.0mol/mol), buffer solution pH (4.0-9.0), reaction temperature (25-45℃), cell addition (12-24g DCW/L), and substrate concentration (50-300g/L).[11]

The conditions for (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one biosynt hesis using smCR125 cells were preliminarily optimized as:100mM NaH2PO4-Na2HPO4 buffer at pH7.0, DMSO concentration of 5% (vol/vol) and the temperature of 40℃. The addition of the cosubstrate, glucose for the double-enzyme coupling reaction has an indispensable effect on GDH-catalyzed coenzyme regeneration. In order to improve the regeneration efficiency of NADPH, as well as the catalytic rate of ET-4, the ratio of glucose to ET-4 (mol/mol) was optimized, ranging from 1.0 to 2.0. The results demonstrated that when the ratio of glucose to ET-4 (mol/mol) was 1.2, the highest yield of 63.4% was achieved after 1 hr reaction. As the ratio further increased, no significant improvement of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one yield was observed. The cell amount for the biotransformation of 150 g/L ET-4 was subsequently investigated. When the cell amount was increased from 14 to 20g DCW/L, the initial reaction rate increased gradually and the yield reached 99.9% after 12 hr reaction and no improvement in the reaction rate was achieved if more cells were added. Considering of the catalyst cost, 20 g DCW/L was chosen as the optimal cell amount.[11]

Factors influencing on the biosynthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one:1.Effects of extra cofactor on the biosynthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one:Nicotinamide cofactors were necessary in the asymmetric reduction of ET-4 to (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one, while the expensive price of exogenous nicotinamide coenzymes increased the cost of biocatalytic process and limited large-scale industrial applications of CRs. In order to evaluate the regeneration efficiency of NADPH in E. coli smCR125, 0 and 0.2 mM cofactor were added to the asymmetric reduction system, and the reaction progress was analyzed. In the first 4 hr, with the extra addition of NADPH, the product yield increased higher than that without the addition of coenzyme. However, at the seventh hr, the yield reached above 99.0% whether extra cofactor was added or not, which indicated that the level of NADPH regeneration in recombinant E. coli cells was adequate during the reaction process and no exogenous NADPH was needed.[11]

2.Biosynthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one under different substrate concentration:Different substrate concentrations for the production of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one were investigated. When the substrate concentration increased from 100 to 200g/L, the yield of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one kept at a high level. As ET-4 concentration further increased to 300 g/L, the product yield decreased gradually to 85.2%. ET-4 (200 g/L) was considered to be the optimal substrate concentration. The detailed reaction process under this concentration was further evaluated. For the first 2 hr, the catalytic rate was fast and the yield of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one reached 60.8%. Then the reaction rate decreased gradually and the product yield reached 94.8% at 10th hr. After 22 hr asymmetric reduction, ET-4 was almost completely transformed to (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoy1]-4-pheny-1,3-oxazolidin-2-one, the (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one yield of 99.8% and the e.e. of 99.9% were finally achieved in the biotransformation process.[11]

3.Effects of ET-4 feeding strategies on the synthesis of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one:The fed-batch strategy for ET-4 addition was constructed to increase the total substrate concentration and to improve the catalytic efficiency for the potential upscale applications. The following four feeding methods were investigated:(a) 250g/L ET-4 and 150g/L glucose were added both once to the reaction system. (b) 125 g/L ET-4 and 75g/L glucose were added to the reaction system twice at 0hr and 2th hr, respectively. (c) 100g/L ET-4 and 60g/L glucose were added at the beginning of the reaction. At 2nd and 4th hr, 75g/L ET-4 and 45g/L glucose were added twice to the reaction system, respectively.(d) 125g/L ET-4 and 150g/L glucose were added at the beginning of the reaction. Then, another 125g/L ET-4 was added at 2nd hr. The results showed that compared with the one -time addition of substrate, the catalytic efficiency of the fed-batch methods improved significantly. Among them, the highest yield of (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one was achieved to 98.9%, and the e.e. was 99.9% with the total substrate concentration of 250g/L. Compared with Scheme 2 and 3, the fed-batch strategy not only relieved the substrate inhibition to CR125 by adding the total ET-4 for twice, but also kept the cofactor regeneration at high level with the initial glucose concentration of 150g/L The reaction details are shown. Without the addition of extra NADPH, under the optimal conditions, 125g/L ET-4 and 150g/L glucose were added at the beginning of the reaction and after 2 hr asymmetric reduction, another 125g/L ET-4 was added to the reaction system. After 12 hr of reaction, ET-4 was almost transformed to (4S)-3-[(5R)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-pheny-1,3-oxazolidin-2-one with a yield of 98.9% and e.e. of 99.9%.[11]

References

[1]Liu Z Q, Dong S C, Yin H H, et al. Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase in an aqueous-organic solvent system[J]. Bioresource Technology, 2017, 229:26.

[2]?nie?ek M, Stecko S, Panfil I, Furman B, Chmielewski M. Total synthesis of ezetimibe, a cholesterol absorption inhibitor[J]. Org Chem. 2013, 78:7048-7057.

[3]Singh A, Goel Y, Rai AK, Banerjee UC. Lipase catalyzed kinetic resolution for the production of (S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one: an intermediate for the synthesis of ezetimibe[J]. J Mol Catal, B Enzym. 2013;85:99-104.

[4]Bertrand B, Durassier S, Frein S, Burgos A. Process for preparing ezetimibe intermediate by enantioselective CBS catalyzed ketone reduction with BH3-DEA prepared in situ. Tetrahedron Lett. 2007;48: 2123-2125.

[5]Ye Q, Ouyang P, Ying H. Biosynthesis of optically pure ethyl (S)- 4-chloro-3-hydroxybutanoate ester: recent advances and future perspectives. Appl Microbiol Biotechnol. 2011;89:513-522.

[6]Hollmann F, Arends IWCE, Holtmann D. Enzymatic reductions for the chemist. Green Chem. 2011;13:2285-2314.

[7]Li M, Zhang ZJ, Kong XD, Yu HL, Zhou JH, Xu JH. Engineering streptomyces coelicolor carbonyl reductase for efficient atorvastatin precursor synthesis. Appl Environ Microbiol. 2017;83:673-678.

[8]Homann MJ, Previte E. Stereoselective microbial reduction of 5-fluorophenyl-5-oxo-pentanoic acid and a phenyloxazolidinone condensation product thereof. US5618707, 1997.

[9]Homann MJ, Previte E. Stereoselective microbial reduction process. EP0862645B1, 2003.

[10]Liu ZQ, Dong SC, Yin HH, et al. Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase in an aqueous-organic solvent system. Bioresour Technol. 2017;229:26-32.

[11]Zhang X, Zhou R, Wu D, et al. Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase[J]. Biotechnology Progress.

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