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Chemoenzymatic synthesis of the chiral side-chain of statins: application of an alcohol dehydrogenase catalysed ketone reduction on a large scale

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Abstract

The chemoenzymatic synthesis of the tert-butyl (S)-6-chloro-5-hydroxy-3-ketohexanoate is described. Our approach relies on a highly regio- and enantioselective reduction of a β,δ-diketohexanoate ester catalysed by NADP(H)-dependent alcohol dehydrogenase of Lactobacillus brevis (LBADH). A detailed description of the scale-up of the enzymatic synthesis of the hydroxyketo ester is given which includes a scale-up of the substrate synthesis as well, i.e. the preparation of diketo ester on a 100 g scale. Furthermore, studies directed towards improving the co-catalyst [NADP(H)] consumption of the enzymatic key step by kinetic studies and application of a biphasic reaction medium were performed.

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Notes

  1. After finishing the work described here, we became aware of several sources that reported about difficulties on attempted preparation of terminally chlorinated β,δ-diketo esters [22, pp 46–47]. Attempts to synthesize methyl 3,5-dioxo-6-chlorohexanoate […] using a literature procedure (Yamaguchi et al. [25]) were unsuccessful. The chloro 3,5-dioxo ester […] remained the most elusive in the syntheses described in this section.”

  2. This is in contrast to the finding of Langer et al., who report failure of this reaction [20, 21].

  3. Attempted acylation under the same conditions (T < −65 °C, 1 eq, bisenolate) using methyl propionate and methyl butanoate as acylation reagents—two esters that do not carry an α-heteroatom substituent—resulted in poor yields (<25%). Large amounts of unreacted tert-butyl acetoacetate were found in the crude products of these reactions. These results are in accordance with the findings of Huckin and Weiler [23, 24]. Presumably, the α-chloro substituent of methyl chloroacetate 14 can stabilise the primary tetrahedral addition intermediate which prevents the harmful liberation of the newly formed keto group prior to hydrolytic work-up. The same protective effect is assigned to the N-methoxy group of Weinreb amides in this kind of ketone synthesis [26–28].

  4. The thermodynamic equilibrium is constant and is determined to be \( K_{{{\text{prod}}}} = \frac{{{\left[ {{\text{NADP}}} \right]}{\left[ \bf 9 \right]}}} {{{\left[ {{\text{NADPH}}} \right]}{\left[ {H^{ + } } \right]}{\left[ {\bf 10} \right]}}} \ge 1.4 \times 10^{8} \) For determination of equilibrium conversion in biphasic catalysed reactions; cf. [29].

  5. In terms of process parameters like pH, temperature, stirring, presence of organic solvent, etc. the requirement of a coupled second enzymatic system for cofactor regeneration can narrow the operational window to an unacceptable extent in difficult cases. In the present case, such a coupled enzyme reaction for cofactor regeneration would have to be compatible not only with the relative low pH, but also with the high load of 2-propanol, the high shear forces of the applied type of mixing, and the presence of the reactive alkylator 10 to name but a few. As an example, we observed strong inhibitory effects of 10 on formate dehydrogenase and metabolic activity of Saccharomyces cerevisiae in another study directed towards enzymatic reduction of 10.

  6. The reverse reaction, i.e. the oxidation of 9–10 is also catalysed by LBADH. At 25 °C and the concentration of 9 being 15 and 2 mM NADP, the activity measured for oxidation is 3.5 U/mgenzyme. This value corresponds to 1% of the standard activity. Given the low reaction rate and equilibrium position under high 2-propanol concentration employed in this work, this reaction can be regarded as less important. Indeed it was seldom observed in the course of experimental work. Therefore it is disconsidered for our purposes.

  7. Nevertheless, determination of V max and the relative activity is not necessary for the fed batch process development.

  8. Similar observations were made in respect to HLADH [35].

  9. For phosphate/citrate buffer solution at pH 5.5 it is 0.0008 h−1.

  10. As reaction volume only the aqueous phase is considered. The organic phase can be regarded as an inert reservoir of substrate. Hence, the real concentration of substrate in the aqueous phase is not increased in the biphasic system [38].

  11. Analytical data of diketo ester 10, hydroxy keto ester 9 and furanone 18 have been published elsewhere [15–17].

  12. Analytical data of diketo ester 10, hydroxy keto ester 9 and furanone 18 have been published elsewhere [15–17].

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Authors and Affiliations

  1. Institute of Biotechnology 2, Research Center Jülich, 52425, Jülich, Germany

    Michael Wolberg, Murillo Villela Filho, Silke Bode, Petra Geilenkirchen, Ralf Feldmann, Andreas Liese & Michael Müller

  2. Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104, Freiburg, Germany

    Murillo Villela Filho, Silke Bode & Michael Müller

  3. Institute of Molecular Enzyme Technology, Heinrich-Heine University Duesseldorf, Research Center Jülich, 52426, Jülich, Germany

    Werner Hummel

  4. Institute of Technical Biocatalysis, Hamburg University of Technology (TUHH), 21073, Hamburg, Germany

    Andreas Liese

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  1. Michael Wolberg
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Correspondence to Michael Müller.

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Dedicated to Prof. C. Wandrey on the occasion of his 65th birthday.

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Wolberg, M., Filho, M.V., Bode, S. et al. Chemoenzymatic synthesis of the chiral side-chain of statins: application of an alcohol dehydrogenase catalysed ketone reduction on a large scale. Bioprocess Biosyst Eng 31, 183–191 (2008). https://doi.org/10.1007/s00449-008-0205-9

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