Improvement of the biocatalytic properties of one phenylacetone monooxygenase mutant in hydrophilic organic solvents

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Abstract

The presence of different hydrophilic organic solvents or a water soluble polymer such as PEG 4000 led to an enhancement in the enzymatic activity of the M446G mutant of phenylacetone monooxygenase when it is employed in enantioselective sulfoxidations and Baeyer–Villiger reactions. By solvent engineering new substrates were found to be effectively converted by this Baeyer–Villiger monooxygenase. The use of 5% methanol together with the weak anion exchange resin Lewatit MP62 also allows the dynamic kinetic resolution of a set of racemic benzylketones. By this approach (S)-benzylesters could be obtained with high yields and optical purities.

Highlights

► The effect of organic solvents on M446G mutant of phenylacetone monooxygenase was examined. ► Presence of hydrophilic solvents led to an increase in the enzymatic activity. ► The use of 5% methanol combined with anion exchange resins allowed DKR processes.

Introduction

Phenylacetone monooxygenase (PAMO, EC 1.14.13.92) from Thermobifida fusca is a monomeric and thermostable Baeyer–Villiger monooxygenase (BVMO), discovered by genome mining in 2005 [1]. BVMOs represent a highly valuable class of oxidoreductases, being able to catalyze a set of interesting reactions in organic synthesis, such as the Baeyer–Villiger oxidation and the oxygenation of different heteroatoms, in general with high selectivities and under mild reaction conditions [2], [3], [4]. PAMO is a well-known BVMO biocatalyst, which structure has been elucidated by X-ray crystallography [5] and its mechanism has been deeply studied [6]. It is active on a wide range of aromatic substrates, while it also accepts some aliphatic compounds. We have shown that this enzyme is able to perform the kinetic resolution of racemic benzylketones with high selectivity [7], [8]. PAMO also oxidizes selectively aromatic sulfides and racemic sulfoxides, as well as nitrogenated compounds and the boron atom [9]. One interesting property of this biocatalyst is its ability to perform oxidative processes while working in non-conventional reaction media [10]. The use of organic cosolvents allows expanding the range of substrates, by working with hydrophobic substrates, and serves to develop solvent engineering techniques to alter the biocatalytic properties of this BVMO. Indeed, we have described that the use of PAMO in the presence of low amounts of short chain alcohols such as methanol or ethanol resulted in significant improvements of enantioselectivity in the oxidation of prochiral sulfides and racemic ketones [11], [12]. Remarkably, for some reactions methanol was able to cause a complete reversal of PAMO enantiopreference. The beneficial effect of organic cosolvents for PAMO-based applications have been also shown in the kinetic resolution of α-acetylphenylacetonitrile, leading to higher conversions and minimizing the amount of undesired side reaction in the presence of different concentrations of EtOAc or iPr2O [12].

Using the PAMO structure, some mutants of this biocatalyst have been engineered [13], [14]. The M446 residue of PAMO has been recently replaced by a glycine [15], after comparing the PAMO structure with a homology-model of cyclopentanone monooxygenase (CPMO, EC 1.14.13.16) [16]. M446G PAMO retains PAMO thermostability and produces an altered substrate binding pocket, which explains the substantial changes in substrate specificity and enantioselectivity towards sulfides and ketones. This biocatalyst was found to be active with a number of aromatic ketones, amines and sulfides for which wild type PAMO shows no activity. Recently it has been employed in the oxidation of benzofused ketones [17], and in the dynamic kinetic resolution of 2-alkyl-1-indanones [18]. It is worth noting that these compounds were no substrates of the wild type enzyme. In addition to altered substrate specificity, the enantioselectivity towards several sulfides was dramatically improved. This newly designed Baeyer–Villiger monooxygenase extends the scope of oxidation reactions feasible with these atypical monooxygenases. For this reason, in this study we report on the activity, stability and selectivity of M446G PAMO when working in the presence of organic cosolvents, with the aim to extend our knowledge on this enzyme and increase its applicability in organic synthesis.

Section snippets

Reagents and biocatalysts

Recombinant histidine-tagged phenylacetone monooxygenase M446G mutant (M446G) was overexpressed and purified as described previously [15]. One unit of M446G will oxidize 1.0 μmol of thioanisole (2a) to methyl phenyl sulfoxide (2b) per minute at pH 9.0 and 25 °C in the presence of NADPH. Glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides were obtained from Fluka-Biochemika. Chemical reactions were monitored by analytical TLC, which was performed

Effect of organic cosolvents in M446G-catalyzed sulfoxidations

The M446G PAMO-biocatalyzed oxidation of benzyl methyl sulfide (1a) was analysed using G6P/G6PDH as secondary ancillary system for regenerating the NADPH cofactor [20]. This compound (1a) has been chosen as model substrate due to the moderate optical purity obtained in its M446G-catalyzed sulfoxidation [15]. The effect of the pH and the temperature on the biocatalyst properties was optimized, which revealed the same pattern as for wild type PAMO. Thus, the improvement of the pH or the

Conclusions

The recently discovered BVMO phenylacetone monooxygenase (PAMO) has attracted our attention due to the special properties acquired by this biocatalyst when performing its activity in non-conventional medium. The properties of its M446G mutant were also modified by the addition of organic solvents, resulting in a different behaviour when compared with the wild type enzyme. For wild type PAMO, short chain alcohols led to a decrease in conversions and higher selectivities, while the presence of

Acknowledgments

A.R.-M. (FPU program) thanks the Spanish Ministerio de Ciencia e Innovación (MICINN) for her predoctoral fellowship. Financial support from MICINN (Project CTQ2007-61126) is gratefully acknowledged.

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