Establishment Of A New(R)-Ethyl-3-Hydroxyglutarate Biosynthesis System Via An Indirect Pathway

As effective cholesterol-lowering drugs, statins are the best-selling drugs in the world market. In particular, the third-generation statin rosuvastatin elicits good response in therapy for patients with coronary artery disease. The3,5-dihydroxyheptanoate side chain derived from (R)-ethyl-3-hydroxyglutarate is the key side chain of rosuvastatin, so the preparation of (R)-3with stereochemical purity has been a controversial issue. The development of a effective, cheap and low pollution technology is significant to reduce the price of rosuvastatin, improve the quality of the product and enhance the international competition of national pharmaceutical company in this field. The present study was aimed to develop an effective biosynthesis method of (R)-ethyl-3-hydroxyglutarate with ethyl (R)-4-cyano-3-hydroxybutyate as substrate.(1) Development of a simple detection method. There was no detection method for the simultaneous determination of (R)-ethyl-4-cyano-3-hydroxybutyate,(R)-ethyl-3-hydroxyglutarate and ethyl (R)-4-carbamoyl-3-hydroxybutanoate, which was so important to determine the reaction pathway. Through analysis of the structures and properties of the three substances, based on the specificness of ionic compounds in the elution of reversed phase high-performance liquid chromatography (HPLC), an HPLC assay for the simultaneous determination of the three compounds was established. According to the optimization of mobile phase compositions and pH values, the optimal conditions were as follows:ODS C18column (250mm×4.6mm,5μm);210nm detection wavelength; mobile phase consisting of methanol and50mM sodium phosphate buffer pH6.5(3:7, v/v);40℃column temperature. By method validation, this method showed good linearity, repeatability, specificity, low limits of detection (LOD) and quantification (LOQ), and short analytical time.(2) Screening targeted strains. By screening two soil samples from Mei Mountain of Jiangsu and Five-finger Mountain of Hainan, some wild strains with low nitrile-hydrolyzing activities were obtained with a selected screening method and a colorimetric screening method. However, the activities could not meet the requirement of the industrial production. Through a high throughpt PCR screening method, two conserved sequences of nitrilase were obtained from mixed bacterial genome. Screening the strains stored at our laboratory, Rhodococcus boritolerans showed good activity on the transformation from (R)-ethyl-4-cyano-3-hydroxybutyate to (R)-ethyl-3-hydroxyglutarate.(3) Determination of the reaction pathway and compound structures. In order to determine the reaction pathway, the time course for the biotransformation was carried out. The results of HPLC detection showed the presence of an intermediate, indicating that the reaction was conducted through an indirect two-step reaction. The indirect reaction pathway was further conformed when (R)-ethyl-3-hydroxyglutarate was obtained by the above reaction using ethyl (R)-4-carbamoyl-3-hydroxybutanoate as substrate. The intermediate and product were both identified by electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) and optical rotation analysis. The above data were identical to those of reported (R)-4-carbamoyl-3-hydroxybutanoate and (R)-ethyl-3-hydroxyglutarate. Therefore, the reaction pathway was finally determined as an indirect pathway with (R)-4-carbamoyl-3-hydroxybutanoate as intermediate, indicating that R. boritolerans harbors potential nitrile hydratase and amidase.(4) Optimization of the transformation conditions. With the optimization of temperature, pH, and substrate concentration, the best conditions were as follows:25℃; pH7.5;10g/1(R)-ethyl-4-cyano-3-hydroxybutyate. Under this condition, the final conversion was98%(w/w), which was the maximum during the reported reaction from (R)-ethyl-4-cyano-3-hydroxybutyate to (R)-ethyl-3-hydroxyglutarate. Additionally, the best substrate concentration was high, so the optimal system has potential applications in the industry production of (R)-ethyl-3-hydroxyglutarate.The present study firstly developed an indirect biosynthesis system of (R)-ethyl-3-hydroxyglutarate with (R)-ethyl-4-cyano-3-hydroxybutyate as substrate and R. boritolerans as the catalyst. This system is low-cost, environment-friendly and has the top conversion during the reported biosynthesis system, which could help release the environmental pressure of chemical drug production.