Research Article: A Highly Productive, Whole-Cell DERA Chemoenzymatic Process for Production of Key Lactonized Side-Chain Intermediates in Statin Synthesis

Date Published: May 7, 2013

Publisher: Public Library of Science

Author(s): Matej Ošlaj, Jérôme Cluzeau, Damir Orkić, Gregor Kopitar, Peter Mrak, Zdenko Časar, Andrew C. Marr. http://doi.org/10.1371/journal.pone.0062250

Abstract

Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L−1 h−1 (up to 50 g L−1 h−1) with >80% yield and >80% chromatographic purity with titers reaching 100 g L−1. Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.

Partial Text

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase [1]–[3], are one of the most pronounced success stories of modern medicinal chemistry [4]. Beside their cholesterol-lowering capabilities, they have been found also to possess many other beneficial effects [5]–[9]. Statins consist of a chiral diol side chain, appended to a cyclic fragment. Initially discovered as microbial metabolites [10]–[13], statins have been rapidly developed into even more efficient synthetic analogues by a partial modification of their structure. The fully synthetic derivatives are frequently addressed as super-statins [14]. Due to the fact that the side chain represents an essential building block in all statins, vast research efforts have been made for its efficient construction and incorporation into the final statin structure. These studies have demonstrated that statins are readily accessible through a variety of different approaches utilizing various types of side-chain precursors. Indeed, statins have been built from open side-chain derivatives, lactol type precursors [14] and very recently from a lactonized side-chain derivative [15], [16]. Due to the increased commercial demand for statins and the need for a simple preparation on industrial scale, impetus for even more efficient, environmentally friendly and easily scalable preparation of the side-chain derivatives appeared. Moreover, synthetically demanding chiral diol structure of the side chain and high quality requirements of pharmaceutical industry on its (stereo)chemical purity shifted the research from initially pure chemical synthesis to enzyme-based approaches, which are known to perform with high stereoselectivity [17]–[32].

The described process for production of chiral, lactonized super-statin intermediates, using a whole-cell DERA catalyst derived directly from a fed-batch, high-density fermentation with E. coli BL21 (DE3) overexpressing native E. coli deoC, shows unprecedented productivity and is highly cost effective. Volumetric productivities of 50 g L−1 h−1, with >80% yield and >80% chromatographic purity have been achieved with this process for 6′-chloro and 6′-acetyloxy substituted lactols. Moreover, ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate and (4R,6S)-6-(dimethoxymethyl)tetrahydro-2H-pyran-2,4-diol have been prepared, using DERA catalysis, for the first time. Although new insights into the dynamics of the reaction intermediates and side products are presented in this work, the complexity of the reaction leaves room for additional improvement of understanding of the DERA-catalyzed sequential aldol condensation reactions. This is especially true for the role of equilibria of the reaction aldehydes with their hydrate form on the one side and acetal/hemiacetal forms on the other. In addition, the dynamics of the availability of the substrates, defined by the feeding strategy and equilibria with their non-reactive forms in combination with substrate preference of the DERA enzyme, can lead the reaction to form completely unexpected products such as 2,6-chloro-2,4-dideoxyhexose. Nevertheless, excellent enantiomeric purities (ee >99.9%) which were demonstrated on downstream advanced intermediates allow the products obtained by the described process to fit directly into our recently described lactone pathway to optically pure super-statins. The enzymatic synthesis of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate is the last piece in design of an efficient and industrially scalable synthesis of super-statins based on the direct coupling of lactonised diol side-chains to the heterocyclic part of the molecule.

Source:

http://doi.org/10.1371/journal.pone.0062250