Alain Archelas

Microbiological transformation 32: Use of epoxide hydrolase mediated biohydrolysis as a way to enantiopure epoxides and vicinal diols: Application to substituted styrene oxide derivatives

The biohydrolyses of various substituted styrene oxide derivatives using the fungi Aspergillus niger or Beauveria sulfurescens are described. The results obtained show that this methodology allows the preparation of enantiomerically enriched epoxides and diols via enantioselective and regioselective hydration. The comparative study of the results obtained suggests that these hydrolyses operate following different mechanisms and a model of the corresponding active sites is proposed.

Microbial Transformations 16. One-step synthesis of a pivotal prostaglandin chiral synthon via a highly enantioselective microbiological Baeyer-Villiger type reaction

Abstract The bioconversion of bicyclo[3.2.0]hept-2-en-6-one is described. Each one of its enantiomers affords a different lactone via a highly regio and enantiospecific Baeyer-Villiger type process.

Microbiological transformations 37. An enantioconvergent synthesis of the β-blocker (R)-Nifenalol® using a combined chemoenzymatic approach

Abstract This work describes the synthesis of (R)- Nif e nalol ® based on an enantioconvergent chemoenzymatic hydrolysis of para-nitrostyrene oxide. A mathematical approach is devised which allowed to optimize the process.

Microbiological transformations. Part 45: A green chemistry preparative scale synthesis of enantiopure building blocks of Eliprodil: elaboration of a high substrate concentration epoxide hydrolase-catalyzed hydrolytic kinetic resolution process

Abstract The enantioselective hydrolysis of racemic para-chlorostyrene oxide 2 following a typical ‘green chemistry’ procedure based on the use of two different Epoxide Hydrolases is described. This allows the preparation of both enantiomers of 2 in very high enantiomeric purity. Furthermore, using a ‘one-pot’ sequential bi-enzymatic strategy enabling to overcome the 50% yield limitation intrinsic to any resolution process, rac-2 could be transformed into nearly enantiopure (R)-3 with an overall yield as high as 93%. The methodology developed was based on the use of a biphasic reactor at high substrate concentration, which is highly desirable for any potential industrial process. The obtained chirons are valuable building blocks for the synthesis of various biologically active targets, like (R)-Eliprodil.

Epoxide hydrolases: new tools for the synthesis of fine organic chemicals

Epoxide hydrolases are ubiquitous enzymes able to hydrolyse an epoxide to its corresponding vicinal diol. These hydrolases have been shown often to be highly enantio- and regioselective, thus allowing both the epoxide and the diol to be prepared at high enantiomeric purity. Because these products show high chemical versatility, they are important for the synthesis of various biologically active products. Recent studies have provided valuable information on the molecular structure of these enzymes, as well as insight to the enzymatic mechanisms involved.

Enantioselective Hydrolysis of p-Nitrostyrene Oxide by an Epoxide Hydrolase Preparation from Aspergillus niger

The epoxide hydrolase activity of Aspergillus niger was synthesized during growth of the fungus and was shown to be associated with the soluble cell fraction. An enzyme preparation was worked out which could be used in place of the whole mycelium as biocatalyst for the hydrolysis of epoxides. The effect of four different cosolvents on enzyme activity was investigated. Consequently, dimethylsulfoxide (DMSO) was selected for epoxide solubilization. The effect of temperature on both reaction rate and enzyme stability was studied in the presence of DMSO (0.2 volume ratio). A temperature of 25°C was selected for the reaction of bioconversion. With a substrate concentration of 4.5 mM a batch reactor showed that the enzyme preparation hydrolyzed para‐nitrostyrene oxide with very high enantioselectivity. The (S) enantiomer of the epoxide remained in the reaction mixture and showed an enantiomeric excess higher than 99%. The substrate concentration could be increased to 20 mM without affecting the enantiomeric excess and degree of conversion. Therefore, the method is potentially useful for the preparative resolution of epoxides. Application are in the field of chiral synthons which are important building blocks in organic synthesis.

Asymmetric hydrolysis of racemic para-nitrostyrene oxide using an epoxide hydrolase preparation from Aspergillus niger

Abstract A lyophilized epoxide hydrolase preparation was isolated from the fungus Aspergillus niger . The preparation could be used in place of the whole mycelium as biocatalyst for the enantioselective hydrolysis of racemic para-nitrostyrene oxide. The cosolvent used for substrate dissolution showed slightly different effects on enzyme activity and stability. Dimethylsulfoxide (DMSO) was selected as the less inhibitory cosolvent among those tested. The enzyme preparation was first proved efficient by running a batch reactor at a low substrate concentration of 4 m m . The hydrolysis of para-nitrostyrene oxide was fast (around 5 h) and with high enantioselectivity ( E = 41). The (S) enantiomer of the epoxide remained in the reaction mixture with an enantiomeric excess (ee) higher than 97% for a conversion of 47%. The substrate concentration had been optimized. It could be increased to 330 m m (54 g l −1 ) without affecting the ee; therefore, the method is potentially useful for the preparative resolution of epoxides. Applications are in the field of chiral synthons which are important building blocks in organic synthesis.

Microbiological transformations. Part 39: Determination of the regioselectivity occurring during oxirane ring opening by epoxide hydrolases: a theoretical analysis and a new method for its determination

Abstract In the course of this work we have devised new equations as well as a new method allowing for the total determination of the regioselectivity occurring during biohydrolysis of a racemic epoxide by an epoxide hydrolase. This determination is achievable by simply studying the racemic epoxide as a substrate. The results showed that, depending on the enantioselectivity (E value) and the regioselectivity involved, the absolute configuration as well as the enantiopurity of the residual epoxide and of the formed diol appear to be highly variable. For a specific enzyme/substrate couple, the yield and enantiopurity of the less reactive (remaining) epoxide—and thus the possibility to prepare it in enantiopure form—exclusively depend upon the enzyme enantioselectivity. On the other hand, the ee of the formed diol (eep) depends upon the enantioselectivity and on the regioselectivity of the oxirane ring opening. A theoretical analysis based on the material balance, as well as several practical examples, are provided to illustrate the various possibilities of such biohydrolyses.

Epoxide Hydrolases and Their Synthetic Applications

Chiral epoxides and 1,2-diols, which are central building blocks for the asymmetric synthesis of bioactive compounds, can be obtained by using enzymes--i.e. epoxide hydrolases--which catalyse the enantioselective hydrolysis of epoxides. These biocatalysis have recently been found to be more widely distributed in fungi and bacteria than previously expected. Sufficient sources from bacteria, such as Rhodococcus and Nocardia spp., or fungi, as for instance Aspergillus and Beauveria spp., have now been identified. The reaction proceeds via an SN2-specific opening of the epoxide, leading to the formation of the corresponding trans-configured 1,2-diol. For the resolution of racemic monosubstituted and 2,2- or 2,3-disubstituted substrates, various fungi and bacteria have been shown to possess excellent enantioselectivities. Additionally, different methods, which lead to the formation of the optically pure product diol in a chemical yield far beyond the 50% mark (which is intrinsic to classic kinetic resolutions), are discussed. In addition, the use of non-natural nucleophiles such as azides or amines provides access to enantiomerically enriched vicinal azido- and amino-alcohols. The synthetic potential of these enzymes for asymmetric synthesis is illustrated with recent examples, describing the preparation of some biologically active molecules.

A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity

In this paper we report the development of a novel and simple spectrophotometric assay which allows one to achieve the continuous, rapid, sensitive, and accurate determination of an epoxide hydrolase activity. This assay is based on the elaboration of a coupled enzymatic/chemical methodology which allows quantification of the enzymatic activity within 3min, and offers good sensitivity of about 10 micro Mmin(-1). Applicability of this test to some other aromatic epoxides has been shown and some limitations have also been explored. This assay should be particularly useful for different applications, for example (a) activity localization during purification of such enzymes, (b) very rapid determination of kinetic constants, and (c) high-throughput screening experiments.