The study on the applications of Cyclohexanone Monooxygenase in Baeyer – Villiger Oxidation

The study on the applications of Cyclohexanone Monooxygenase in Baeyer – Villiger Oxidation

Silesian University of Technology, Department of Chemical Organic Technology and Petrochemistry, ul. Krzywoustego 4, 44-100 Gliwice, Poland

Agnieszka Drożdż, Anna Chrobok

RESULTS

This work presents study on the cloning, expression and purification of Cyclohexanone monooxygenase Acinetobacter sp. in prokaryotic expression system Escherichia coli and the application of this enzyme in BV oxidation of cyclic ketones to the chiral lactones.

In the first part of the study, gene chnB encoding Cyclohexanone Monooxygenase was cloned from genomic DNA isolated from the strain Acinetobacter johnsonii NCIMB9871. For efficient expression of CMO the pGEX_5T expression vector was selected. The sequencing of DNA confirmed the formation of construct pGEX_5T_chnB in expression system Escherichia coli, which allows the expression of proteins with CMO GST fusion tag.

As a result 20 mg of the desired protein Cyclohexanone Monooxygenase diluted in phosphate buffered saline (PBS) (concentration 1.2 mg/ml) was obtained. High purity of protein (95%) was confirmed by electrophoresis (Fig. 2). The enzymatic activity 11 U/mg was determined by the observation of the decreasing of the amount of NADPH which is oxidized to NADP in the presence of cyclohexanone and protein (see Figure 3). This research was conducted in the co-operation with BioCentrum company (Kraków).

INTRODUCTION

The Baeyer-Villiger (BV) reaction is based on the formation of esters and lactones by the oxidation of ketones with peroxide derivatives. It is still one of the most important reactions in organic chemistry, with a large range of possible applications, including the synthesis of antibiotics, steroids, pheromones, and monomers for polymerization. Biocatalytic version of this reaction is based on the application of the so-called Baeyer-Villiger monooxygenases (BVMOs) which are extremely promising catalysts useful for enantioselective oxidation of ketones. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIB 9871 is classified as a type I BVMO; this means that it contains flavine adenine dinucleotide (FAD) as a cofactor and uses NADPH as source of electrons. The mechanism of BV reaction with CHMO is shown in Figure 1. In the resting enzyme, non-covalently bound FAD is in its oxidized form and has to be reduced by NADPH to enzyme–NADP⁺ complex, which is then oxidized by molecular oxygen to the flavin peroxide. In the presence of a ketone, nucleophilic attack on the carbonyl group by flavin peroxide leads to the formation of Criegee intermediate, which rearranges to lactone and flavin hydroxide. The elimination of water followed by release of the product and NADP⁺ complete the cycle. The released NADP⁺ must be reduced (outside the enzyme) to return as NADPH for the next turnover.

FIGURE 2.

Electropherogram

representing the purified protein Cyclohexanone Monooxygenase:

1(2 ug), 2(5 mg), 3(10 mg).

M-molecular weight standards in kDa.

FIGURE 3.

The conversion of NADPH to

NADP in time after the addition of cyclohexanone and protein

Cyclohexanone Monooxygenase.

In the next step, the optimization of BV oxidation of model cyclic ketone 4-methylcyclohexanone with oxygen, CHMO and NADPH carried out in in Tris/HCl-buffer (pH 8.0) was performed. For cofactor regeneration two enzymes systems were used: glucose-6-phosphate/glucose 6-phosphate dehydrogenase (G6PDH) or sodium formate/formate dehydrogenase (FDH) (Figure 4). The optimal concentration of all reagents was determined. Conversions were carried out typically on a 1 ml scale (Table 1). The optical purity of lactone was analysed with gas chromatography on chiral column Astec CHIRALDEXTM G-TA.

N

N

NH N

R

O

O

N

N H

NH N^-

O

O R

N

N H

NH N

O R

O O^-

O

N

N H

NH N

O

O R

O OH N

N H

NH N

O

O R

O

O O^-

N

N H

NH N^-

O

O R

OH

O

O O

H₂O

Criegee adduct

FIGURE 1. Proposed mechanism for cyclohexanone monooxygenase- catalyzed Baeyer-Villiger oxidation of cyclohexanone

O

O O

*

CHMO

O₂ ^H2O

NADPH NADP⁺

FDH

CO₂

(G6PDH)

alternative regeneration system:

FIGURE 4. Enzymatic Baeyer-Villiger oxidation with enzyme-coupled cofactor regeneration.

entry CHMO [U]

ketone [mmol/L]

NADPH [mmol/L]

Cofactor regeneration system

ee [%]^a

Yield [%]^b

1 2.4 37 6.0

269mmol/L / 9.6U

62

2 2.4 40 6.0

260mmol/L / 14.4U

93

3 1.2 36 3.0

223mmol/L / 2.4U

13 4 2.0 37 6.0

43 5 2.7 27 0.6

83 6 1.3 22 1.8

99 7 1.3 28 1.8

99 8 1.3 27 0.3

100

99

TABLE 1. Optimalization of concentrations of reagents on the model reaction oxidation of 4-methylcyclohexanone by CHMO, shown in figure 4. The reaction was carried out at 1 ml scale in the Trizma® hydrochloride buffer (pH=8.0) of 24 h in room temperature.

a determined by chiral GC, ^b measured by GC.

In this work, for the first time the application of a CHMO in Baeyer-Villiger reaction with the addition of ionic liquids was reported. In table 2 the preliminary study on the oxidation of 4-methylcyclohexanone with selected ionic liquids was presented. The study concerning the utilization of ionic liquids for enzymatic Baeyer-Villiger reaction will be continue in our laboratory.

Literature:

1. Pazmino D., Dudek H., Fraaije M.: Current Opinion in Chemical Biology, 2010, 14,138–144.

2. Sheng D., Ballou D., Massey V.: Biochemistry 2001, 40, 11156-11167.

3. Clouthier Ch., Kayser M., Reetz M.: J. Org. Chem. 2006, 71, 8431-8437.

4. Mihovilovic M.: Current Organic Chemistry, 2006, 10, 1265-1287.

entry Ionic liquids ee

[%]^a

Yield [%]^b

1 ^H3^C _N⁺ _N CH₃ P O O^-

O

O

CH₃ C

H₃

100 14

2

N N⁺

C H₃

CH₃ PF₆^-

100 43

3

N N⁺

C H₃

CH₃

(CF₃SO₂)₂N^-

100 24

TABLE 2. The effect of 10% of ILs in 50mM Tris/HCl buffer pH 8.0 on the CHMO-catalysed oxidation in appropriate reaction conditions as entered in table 1. entry 8.

CONCLUSION

In summary, was reported en efficient method for cloning, expression and purification of Cyclohexanone monooxygenase and appropriate conditions for BV oxidation. The present study demonstrated for the first time that CHMO can be employed as oxidative biocatalysts in non-conventional media containing ionic liquids in different extents.

Acknowledgement:

This work was financially supported by the Polish State Committee for Scientific Research (Grant no N N209 021739).

Support for the project associated with the competition, "SWIFT’’