P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules

: A set of dual functional small molecules (DFSMs) containing di ﬀ erent amino acids has been synthesized and employed together with three di ﬀ erent variants of the cytochrome P450 monooxygenase P450BM3 from Bacillus megaterium in H 2 O 2 -dependent oxidation reactions. These DFSMs enhance P450BM3 activity with hydrogen peroxide as an oxidant, converting these enzymes into formal peroxygenases. This system has been employed for the catalytic epoxidation of styrene and in the sulfoxidation of thioanisole. Various P450BM3 variants have been evaluated in terms of activity and selectivity of the peroxygenase reactions. synthesized P450BM3-catalyzed oxidations.

The catalytic cycle of P450 monooxygenases comprises the reductive activation of molecular oxygen to form the catalytically active oxyferryl species (i.e., a highly oxidized iron-oxo-complex). The reducing equivalents needed for this reaction are generally derived from reduced nicotinamide cofactors via more or less complex electron transport chains [8], adding complexity to the reaction schemes [9].
In 1999, Arnold and coworkers proposed to preparatively exploit the well-known hydrogen peroxide shunt pathway [10]. Here, the catalytically active compound is formed directly from H 2 O 2 thereby drastically simplifying the regeneration scheme of P450 monooxygenases (Scheme 1).
Unfortunately, the majority of the known P450s are rapidly inactivated by H 2 O 2 making the H 2 O 2 shunt pathway practically irrelevant. Some exceptions are known, in which P450s can efficiently use H 2 O 2 through a substrate-assisted reaction mechanism for the hydroxylation or decarboxylation of fatty acids [11][12][13][14][15].
inactivation of P450 monooxygenases [16,17]. By comparing the catalytic mechanism and active sites of P450 monooxygenases with those of (H2O2-dependent) peroxygenases, these authors reasoned that

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To alleviate this shortcoming, a range of base-modified decoy molecules was suggested. In 55 essence, these dual functional small molecules (DFSMs) comprise an imidazole-base coupled via a 56 linker moiety to an amino acid anchoring part in order to position the base within the P450 57 monooxygenases' active sites, thereby enabling peroxygenase-like reactions [18,19]. In the current 58 study, we set out to validate and broaden this very interesting concept.   Imidazole-based dual DFSMs were synthesized following a literature-known four-step 62 procedure [17,20]. Overall, seven DFSMs comprising different amino acids and different spacer   Recently, Cong and coworkers reported an elegant possible solution to the H 2 O 2 -related inactivation of P450 monooxygenases [16,17]. By comparing the catalytic mechanism and active sites of P450 monooxygenases with those of (H 2 O 2 -dependent) peroxygenases, these authors reasoned that a base (Glutamate) present in peroxygenases but missing in the active site of P450 monooxygenases may account for the poor activity of P450 monooxygenases with H 2 O 2 (Scheme 2).

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Recently, Cong and coworkers reported an elegant possible solution to the H2O2-related 46 inactivation of P450 monooxygenases [16,17]. By comparing the catalytic mechanism and active sites of P450 monooxygenases with those of (H2O2-dependent) peroxygenases, these authors reasoned that

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To alleviate this shortcoming, a range of base-modified decoy molecules was suggested. In 55 essence, these dual functional small molecules (DFSMs) comprise an imidazole-base coupled via a 56 linker moiety to an amino acid anchoring part in order to position the base within the P450 57 monooxygenases' active sites, thereby enabling peroxygenase-like reactions [18,19]. In the current 58 study, we set out to validate and broaden this very interesting concept.    To alleviate this shortcoming, a range of base-modified decoy molecules was suggested. In essence, these dual functional small molecules (DFSMs) comprise an imidazole-base coupled via a linker moiety to an amino acid anchoring part in order to position the base within the P450 monooxygenases' active sites, thereby enabling peroxygenase-like reactions [18,19]. In the current study, we set out to validate and broaden this very interesting concept.

Preparation of the Dual Functional Small Molecules (DFSMs)
Imidazole-based dual DFSMs were synthesized following a literature-known four-step procedure [17,20]. Overall, seven DFSMs comprising different amino acids and different spacer lengths were synthesized (Scheme 3). It should be mentioned here that amongst the seven DFSMs synthesized only 3 (Im-C5-Ile, Im-C6-Phe and Im-C6-Ile) showed significant activity with the enzyme tested.
For the P450 monooxygenase we chose the well-known CYP102A1 (P450BM3) from Bacillus megaterium. More specifically, three variants P450BM3 F87A, P450BM3 V78A/F87A and P450BM3 A74E/F87V/P386S were recombinantly expressed in Escherichia coli and purified following literature methods [21,22]. All variants contained a mutation at position 87, which had previously been reported to broaden the substrate scope of P450BM3 [23]. The side-chain of phenylalanine 87 extends into the lumen of the substrate access channel close to the heme iron and thus residues with less bulky side-chains, such as mutations to alanine or valine, widen the access channel by creating incremental space in the vicinity of the heme iron [23]. The mutation V78A has a similar effect, making the hydrophobic pocket that encloses the heme iron more capacious than in the wild type [23]. The variant P450BM3 A74E/F87V/P386S has previously been shown to possess 2 or 2.5 fold increased catalytic activity for the oxidation of β-ionone compared to the F87A or F87V single variants, respectively, and was therefore also included here [21]. megaterium. More specifically, three variants P450BM3 F87A, P450BM3 V78A/F87A and P450BM3 68 A74E/F87V/P386S were recombinantly expressed in Escherichia coli and purified following literature 69 methods [21,22]. All variants contained a mutation at position 87, which had previously been 70 reported to broaden the substrate scope of P450BM3 [23]. The side-chain of phenylalanine 87 extends 71 into the lumen of the substrate access channel close to the heme iron and thus residues with less bulky 72 side-chains, such as mutations to alanine or valine, widen the access channel by creating incremental space in the vicinity of the heme iron [23]. The mutation V78A has a similar effect, making the 74 hydrophobic pocket that encloses the heme iron more capacious than in the wild type [23]. The 75 variant P450BM3 A74E/F87V/P386S has previously been shown to possess 2 or 2.5 fold increased 76 catalytic activity for the oxidation of β-ionone compared to the F87A or F87V single variants, 77 respectively, and was therefore also included here [21].

Biocatalytic Transformations Using the DFSMs/P450BM3 System
Having all catalytic components at hand, we first investigated the influence of the DFSMs on the P450BM3-catalyzed and H 2 O 2 -driven epoxidation of styrene (1) to obtain optically active styrene oxide (2). As shown in Table 1, only three of the seven DFSMs enabled H 2 O 2 -driven reactions with P50BM3.
Pleasingly, we found that the presence of DFSMs significantly improved the catalytic performance of all P450BM3 variants. In case of the F87A variant for example, Im-C6-Phe increased the product formation almost 20 fold. Other combinations gave similar improvements. However, at present time we are unable to rationalize the improvements in light of DFSM binding to the enzyme active site and/or positioning of the substrates. Further studies will be necessary to obtain a quantitative structure-activity relationship. In line with the pH optimum of P450BM3 [24], the highest turnover numbers were observed at slightly alkaline pH values (Table 1, entries 1 vs. 5 and 6; entries 9 vs. 12). Decreasing the H 2 O 2 concentration appeared to have a positive effect on the product formation (Table 1, entries 1 vs. 7), which we attribute to a lower inactivation rate at lower peroxide concentrations.
Interestingly, the DFSMs also influenced the enantioselectivity of the epoxidation reaction, which is in line with the original report by Cong and coworkers [17]. Possibly, this is due to a more stringent positioning of the starting material in the enzyme active site. However, again, no obvious structure-activity relationship was observed.

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Interestingly, the DFSMs also influenced the enantioselectivity of the epoxidation reaction,

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which is in line with the original report by Cong and coworkers [17]. Possibly, this is due to a more  Similarly, P450BM3-catalyzed sulfoxidation of thioanisole (3) was positively influenced by DFSMs (Table 2). Compared to the epoxidation reaction, rate accelerations were somewhat lower; the enantioselectivity of the sulfoxidation reaction, however, was significantly improved by the DFSMs. Both observations can be rationalized by the spontaneous (non-enantioselective) oxidation of thioanisole by H 2 O 2 [25]. Quite interestingly, the P450BM3 A74E/F87V/P386S variant, which in the epoxidation reaction gave rather poor results compared to the other two variants, excelled in the sulfoxidation reaction.
As mentioned above, H 2 O 2 -related inactivation of the heme enzyme appeared to be a major limitation of the proposed H 2 O 2 -shunt pathway reaction of P450BM3. We therefore also investigated the effect of controlled in situ H 2 O 2 generation via reductive activation of O 2 using an oxidase [26]. Thus, employing the commercially available alcohol oxidase from Pichia pastoris (PpAOx), H 2 O 2 was generated in situ from O 2 at the expense of methanol (which was oxidized to formaldehyde).
When this system was applied (Table 3), reaction rates were significantly decreased (reaction times 18 h), while at the same time the turnover numbers of the biocatalyst were improved, compared to the use of H 2 O 2 they were five times greater. The low concentration of H 2 O 2 available slowed down both the reaction rate and the oxidative inactivation. We expect that further optimized reaction schemes may provide optimal H 2 O 2 generation rates, ensuring maximized enzymatic sulfoxidation while minimizing the H 2 O 2 -related inactivation of the heme enzyme. Again, in the absence of any DFSM, near racemic product was observed, indicating predominant spontaneous sulfoxidation.

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As mentioned above, H2O2-related inactivation of the heme enzyme appeared to be a major 125 limitation of the proposed H2O2-shunt pathway reaction of P450BM3. We therefore also investigated 126 the effect of controlled in situ H2O2 generation via reductive activation of O2 using an oxidase [26].

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Thus, employing the commercially available alcohol oxidase from Pichia pastoris (PpAOx), H2O2 was 128 generated in situ from O2 at the expense of methanol (which was oxidized to formaldehyde).

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When this system was applied (Table 3), reaction rates were significantly decreased (reaction    Determined by Gas Chromatography.

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One major drawback of classic P450 monooxygenation reactions is that, due to the exclusive 145 water solubility of the nicotinamide cofactors, they have to be performed in aqueous reaction media.

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As the majority of the reagents of interest for P450 monooxygenase-catalyzed oxyfunctionalizations 147 are rather hydrophobic, reagent concentrations tend to be in the lower millimolar range, reducing the 148 preparative attractiveness of these reactions from an economic and environmental point-of-view [27].

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In this respect, the proposed peroxide-driven reaction offers some interesting possibilities for non-150 aqueous reactions using P450 monooxygenases.

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To test this hypothesis, we evaluated the epoxidation of styrene using precipitated P450BM3

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F87A suspended in neat styrene as the reaction medium; the stoichiometric oxidant was t BuOOH
One major drawback of classic P450 monooxygenation reactions is that, due to the exclusive water solubility of the nicotinamide cofactors, they have to be performed in aqueous reaction media. As the majority of the reagents of interest for P450 monooxygenase-catalyzed oxyfunctionalizations are rather hydrophobic, reagent concentrations tend to be in the lower millimolar range, reducing the preparative attractiveness of these reactions from an economic and environmental point-of-view [27]. In this respect, the proposed peroxide-driven reaction offers some interesting possibilities for non-aqueous reactions using P450 monooxygenases.
To test this hypothesis, we evaluated the epoxidation of styrene using precipitated P450BM3 F87A suspended in neat styrene as the reaction medium; the stoichiometric oxidant was t BuOOH (Scheme 4).
water solubility of the nicotinamide cofactors, they have to be performed in aqueous reaction media.

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Unless otherwise noted, analytical grade solvents and commercially available reagents were 164 used without further purification.

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Dual functional small molecules (DFSMs) were synthesized according to the methodology 166 described in the literature [17]. Compounds Im-C5-Ile, Im-C5-Phe, Im-C6-Ile, Im-C6-Phe and Im-C6-imidazole. The enzyme was then desalted with a PD-10 column and concentrated with an Amicon filter with a cut-off of 30 kDa.

General Procedure for the Preparation of Im-C4-Phe and Im-C4-Ile
Im-C4-Phe and Im-C4-Ile were prepared starting from 4-(1H-imidazol-1-yl)butanoic acid: A DMF (Dimethylformamide) solution (10 mL) containing HOBt (150 mg, 1.1 mmol), EDC (170 mg, 1.1 mmol), and 4-(1H-imidazol-1-yl)butanoic acid (154 mg, 1.0 mmol) was stirred at room temperature for 1 h. A solution of L-phenylalanine methyl ester or L-isoleucine methyl ester (1.1 mmol) and 4-methylmorpholine (202 mg, 2.0 mmol) dissolved in 10 mL of DMF was then added to the reaction mixture. After 18 h, the reaction mixture was partitioned between dichloromethane (50 mL) and H 2 O (50 mL). The organic layer was washed with H 2 O (3 × 50 mL) and dried over MgSO 4 . The solution was concentrated under reduced pressure. The crude product was then dissolved in 2 mL NaOH aqueous solution (1.0 M) and 1 mL THF and stirred overnight. The THF was removed under reduced pressure and the solution was acidified to pH 2.0 with HCl (1.0 M). Water was then removed under reduced pressure and the residue was dissolved in ethanol. NaCl was separated by filtration and ethanol was evaporated to give the final products.

General Procedure for the Biocatalyzed Oxidation of Styrene and Thioanisole Employing the P450BM3/DFSM System
Unless otherwise stated, the corresponding variant of P450BM3 (0.5 µM) was transferred to a glass sample bottle containing 0.1 M, pH 8.0 phosphate buffer (0.36 mL), styrene (1) or thioanisole (3) (4 mM in methanol) and the DFSM (0.5 mM, dissolved in pH 8.0 phosphate buffer). H 2 O 2 (20 mM, dissolved in pH 8.0 phosphate buffer) was added and the reaction was shaken at room temperature and 300 rpm for 30 min. The reaction was then extracted using ethyl acetate containing 5.0 mM of dodecane as the external standard (0.4 mL) and dried over anhydrous sodium sulfate. The conversion and the optical purity of styrene oxide (2) or methyl phenyl sulfoxide (4) was analyzed by gas chromatography.

General Method for the Biocatalyzed Oxidations Employing the H 2 O 2 In Situ Generation System
P450BM3 F87A (0.5 µM) was transferred to a glass sample bottle containing 0.1 M, pH 8.0 phosphate buffer (0.36 mL), methanol (100 mM), thioanisole (3) (4 mM in methanol) and the DFSM (0.5 mM, dissolved in pH 8.0 phosphate buffer). A solution of the alcohol oxidase from Pichia pastoris (5 nM, dissolved in pH 8.0 phosphate buffer) was added and the reaction was shaken at room temperature and 300 rpm for 18 h. The reaction was then extracted using ethyl acetate containing 5 mM of dodecane as the external standard (0.4 mL) and dried over anhydrous sodium sulfate. The conversion and the optical purity of methyl phenyl sulfoxide (4) was analyzed by gas chromatography.

General Method for the Epoxidation of Styrene Using Precipitated P450BM3 F87A with DFSMs
P450BM3 F87A was precipitated with acetone and dried (30 mg) and transferred to a glass sample bottle containing styrene (1) (200 µL) and the DFSM (0.5 mM, dissolved in pH 8.0 phosphate buffer). t BuOOH (20 mM, 70% in H 2 O) was added and the reaction was shaken at room temperature and 300 rpm for 30 min. The reaction was then extracted using ethyl acetate containing 5.0 mM of dodecane as the external standard (0.4 mL) and dried over anhydrous sodium sulfate. The conversion and the optical purity of styrene oxide (2) was analyzed by gas chromatography.

Conclusions
Overall, we have confirmed Cong's approach, turning P450 monooxygenases into peroxygenases by using DFSMs. The results shown in this study suggest specific interactions of the DFSMs with the enzymes (here P450BM3) influencing their performance as co-catalysts. Further studies with a broader set of DFSMs will be necessary to establish quantitative structure-activity relationships and further optimize the reaction system. It will also be interesting to investigate possible match/mismatch combinations of the (chiral) amino acid anchoring groups.
One exciting possibility of DFSMs arises from the fact that P450 monooxygenase catalysis becomes independent from (exclusively water soluble) nicotinamide cofactors and thereby enables the use of P450 monooxygenases under neat reaction conditions.