P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules

catalysts Communication P450BM3-Catalyzed Oxidations Employing Dual Functional Small Molecules Sébastien J.-P. Willot 1, Florian Tieves 1, Marco Girhard 2, Vlada B. Urlacher 2, Frank Hollmann 1,* and Gonzalo de Gonzalo 3,* 1Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands 2Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany 3Departamento de Química Orgánica, Universidad de Sevilla, c/Profesor García González 1, 41012 Sevilla, Spain *Correspondence: F[email protected] (F.H.); [email protected] (G.d.G.); Tel.: +31-15-2781957 (F.H.); +34-954-559997 (G.d.G.) Received: 5 June 2019; Accepted: 22 June 2019; Published: 26 June 2019   Abstract: A set of dual functional small molecules (DFSMs) containing different amino acids has been synthesized and employed together with three different 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. Keywords: biocatalysis; cytochrome P450; dual functional small molecules; epoxidations; sulfoxidation 1. Introduction Cytochrome P450 monooxygenases (P450 or CYP) catalyze a broad range of oxyfunctionalization reactions of non-activated C–H- and C=C-bonds [ 1 – 6 ]. Especially, the frequently observed regio- and enantioselectivity of this transformation makes P450s potentially very useful tools in preparative biocatalysis [7]. 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 [11–15]. Catalysts 2019,9, 567; doi:10.3390/catal9070567 www.mdpi.com/journal/catalysts Catalysts 2019,9, 567 2 of 9 Molecules 2019, 24, x FOR PEER REVIEW 2 of 10 39 Scheme 1. Comparison of the classical regeneration and the H2O2-shunt pathway to drive P450 40 monooxygenase-catalyzed oxyfunctionalization reactions. 41 Unfortunately, the majority of the known P450s are rapidly inactivated by H2O2 making the H2O2 42 shunt pathway practically irrelevant. Some exceptions are known, in which P450s can efficiently use 43 H2O2 through a substrate-assisted reaction mechanism for the hydroxylation or decarboxylation of 44 fatty acids [11–15]. 45 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 47 of P450 monooxygenases with those of (H2O2-dependent) peroxygenases, these authors reasoned that 48 a base (Glutamate) present in peroxygenases but missing in the active site of P450 monooxygenases 49 may account for the poor activity of P450 monooxygenases with H2O2 (Scheme 2). 50 51 Scheme 2. Formation of Compound I from H2O2 in peroxygenases. The active-site base glutamate 52 (Glu, green) facilitates the reaction by first deprotonating the primary H2O2-adduct and by 53 reprotonation of the peroxo-intermediate. 54 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. 59 2. Results 60 2.1. Preparation of the Dual Functional Small Molecules (DFSMs) 61 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 63 lengths were synthesized (Scheme 3). It should be mentioned here that amongst the seven DFSMs 64 synthesized only 3 (Im-C5-Ile, Im-C6-Phe and Im-C6-Ile) showed significant activity with the enzyme 65 tested. 66 Scheme 1. Comparison of the classical regeneration and the H 2 O 2 -shunt pathway to drive P450 monooxygenase-catalyzed oxyfunctionalization reactions. 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 H2O2(Scheme 2). Molecules 2019, 24, x FOR PEER REVIEW 2 of 10 39 Scheme 1. Comparison of the classical regeneration and the H2O2-shunt pathway to drive P450 40 monooxygenase-catalyzed oxyfunctionalization reactions. 41 Unfortunately, the majority of the known P450s are rapidly inactivated by H2O2 making the H2O2 42 shunt pathway practically irrelevant. Some exceptions are known, in which P450s can efficiently use 43 H2O2 through a substrate-assisted reaction mechanism for the hydroxylation or decarboxylation of 44 fatty acids [11–15]. 45 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 47 of P450 monooxygenases with those of (H2O2-dependent) peroxygenases, these authors reasoned that 48 a base (Glutamate) present in peroxygenases but missing in the active site of P450 monooxygenases 49 may account for the poor activity of P450 monooxygenases with H2O2 (Scheme 2). 50 51 Scheme 2. Formation of Compound I from H2O2 in peroxygenases. The active-site base glutamate 52 (Glu, green) facilitates the reaction by first deprotonating the primary H2O2-adduct and by 53 reprotonation of the peroxo-intermediate. 54 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. 59 2. Results 60 2.1. Preparation of the Dual Functional Small Molecules (DFSMs) 61 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 63 lengths were synthesized (Scheme 3). It should be mentioned here that amongst the seven DFSMs 64 synthesized only 3 (Im-C5-Ile, Im-C6-Phe and Im-C6-Ile) showed significant activity with the enzyme 65 tested. 66 Scheme 2. Formation of Compound I from H 2 O 2 in peroxygenases. The active-site base glutamate (Glu, green) facilitates the reaction by first deprotonating the primary H 2 O 2 -adduct and by reprotonation of the peroxo-intermediate. 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. 2. Results 2.1. 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 Catalysts 2019,9, 567 3 of 9 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]. Molecules 2019, 24, x FOR PEER REVIEW 3 of 10 For the P450 monooxygenase we chose the well-known CYP102A1 (P450BM3) from Bacillus 67 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 73 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]. 78 Br CO2Et 0,1,2 N Na N N NCO2Et 0,1,2 N NCO2H 0,1, 2 NaOH HCl, reflux Toluene, reflux N NCO2H 0,1,2 O NEDC / HOBT DMF, rt, 18h CO2MeR NH2 + N N 0,1,2 H N O CO2Me R NaOH THF / H2O N N 0,1,2 H N O CO2H R 20 - 40% yield 29 - 49% yield NNN H O CO2H NNN H O CO2H Im-C4-Phe, 45% yield Im-C4-Ile, 49% yield NN H O CO2H N NN H O CO2H N N H O CO2H N NN H O CO2H S N N N H O CO2H N N Im-C5-Phe, 37% yield Im-C5-Ile, 29% yield Im-C6-Phe, 37% yield Im-C6-Ile, 46% yield Im-C6-Met, 34% yield 79 Scheme 3. Dual Functional Small Molecules (DFSMs) synthesized for the P450BM3-catalyzed 80 oxidations. 81 2.2. Biocatalytic Transformations Using the DFSMs/P450BM3 System 82 Having all catalytic components at hand, we first investigated the influence of the DFSMs on the 83 P450BM3-catalyzed and H2O2-driven epoxidation of styrene (1) to obtain optically active styrene 84 oxide (2). As shown in Table 1, only three of the seven DFSMs enabled H2O2-driven reactions with 85 P50BM3. 86 87 88 89 Scheme 3. Dual Functional Small Molecules (DFSMs) synthesized for the P450BM3-catalyzed oxidations. 2.2. 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 Catalysts 2019,9, 567 4 of 9 stringent positioning of the starting material in the enzyme active site. However, again, no obvious structure–activity relationship was observed. Table 1. Epoxidation of styrene (1) catalyzed by DFSM/P450BM3 using H2O2as the oxidant.a. Molecules 2019, 24, x FOR PEER REVIEW 4 of 10 Table 1. Epoxidation of styrene (1) catalyzed by DFSM/P450BM3 using H2O2 as the oxidant.a. 90 91 92 Entry DFSM pH c(2) (µM) ee (%)b F87A variant 1 Im-C6-Phe 8 117 72 2 Im-C6-Ile 8 94 59 3 Im-C5-Ile 8 58 54 4 Nonec 8 6 10 5 Im-C6-Phe 7 68 65 6 Im-C6-Phe 6 ≤2 -- 7 Im-C6-Phed 8 133 77 V78A/F87A variant 8 Im-C6-Phe 8 54 6 9 Im-C6-Ile 8 97 22 10 Im-C5-Ile 8 80 19 11 None 8 11 n.d. 12 Im-C6-Ile 6 ≤2 n.d. A74E/F87V/P386S variant 13 Im-C6-Phe 8 ≤2 n.d. 14 Im-C6-Ile 8 17 4 (S) 15 Im-C5-Ile 8 17 6 (S) 16 None 8 ≤2 n.d. a Reaction conditions: P450BM3 (0.5 μM), styrene (4 mM), H2O2 (20 mM; except entry 7), DFSM 93 (0.5 mM), in phosphate buffer. Reaction time: 30 minutes; b Determined by Gas Chromatography; 94 c No reaction was observed in absence of DFSM or biocatalyst; d c(H2O2) = 5 mM; n.d. not 95 determined. 96 Pleasingly, we found that the presence of DFSMs significantly improved the catalytic 97 performance of all P450BM3 variants. In case of the F87A variant for example, Im-C6-Phe increased 98 the product formation almost 20 fold. Other combinations gave similar improvements. However, at 99 present time we are unable to rationalize the improvements in light of DFSM binding to the enzyme 100 active site and/or positioning of the substrates. Further studies will be necessary to obtain a 101 quantitative structure–activity relationship. In line with the pH optimum of P450BM3 [24], the 102 highest turnover numbers were observed at slightly alkaline pH values (Table 1, entries 1 vs. 5 and 103 6; entries 9 vs. 12). Decreasing the H2O2 concentration appeared to have a positive effect on the 104 product formation (Table 1, entries 1 vs. 7), which we attribute to a lower inactivation rate at lower 105 peroxide concentrations. 106 Interestingly, the DFSMs also influenced the enantioselectivity of the epoxidation reaction, 107 which is in line with the original report by Cong and coworkers [17]. Possibly, this is due to a more 108 stringent positioning of the starting material in the enzyme active site. However, again, no obvious 109 structure–activity relationship was observed. 110 Similarly, P450BM3-catalyzed sulfoxidation of thioanisole (3) was positively influenced by 111 DFSMs (Table 2). Compared to the epoxidation reaction, rate accelerations were somewhat lower; the 112 enantioselectivity of the sulfoxidation reaction, however, was significantly improved by the DFSMs. 113 Both observations can be rationalized by the spontaneous (non-enantioselective) oxidation of 114 thioanisole by H2O2 [25]. Quite interestingly, the P450BM3 A74E/F87V/P386S variant, which in the 115 epoxidation reaction gave rather poor results compared to the other two variants, excelled in the 116 sulfoxidation reaction. 117 Entry DFSM pH c(2) (µM) ee (%) b F87A variant 1 Im-C6-Phe 8 117 72 2Im-C6-Ile 8 94 59 3Im-C5-Ile 8 58 54 4None c8 6 10 5Im-C6-Phe 7 68 65 6Im-C6-Phe 6 ≤2 – 7Im-C6-Phe d8 133 77 V78A/F87A variant 8Im-C6-Phe 8 54 6 9Im-C6-Ile 8 97 22 10 Im-C5-Ile 8 80 19 11 None 8 11 n.d. 12 Im-C6-Ile 6 ≤2 n.d. A74E/F87V/P386S variant 13 Im-C6-Phe 8 ≤2 n.d. 14 Im-C6-Ile 8 17 4 (S) 15 Im-C5-Ile 8 17 6 (S) 16 None 8 ≤2 n.d. a Reaction conditions: P450BM3 (0.5 µ M), styrene (4 mM), H 2 O 2 (20 mM; except entry 7), DFSM (0.5 mM), in phosphate buffer. Reaction time: 30 min; b Determined by Gas Chromatography; c No reaction was observed in absence of DFSM or biocatalyst; dc(H2O2)=5 mM; n.d. not determined. 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 O2at 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. Catalysts 2019,9, 567 5 of 9 Table 2. P450BM3-catalyzed sulfoxidation of thioanisole (3) using H2O2as the oxidant.a. Molecules 2019, 24, x FOR PEER REVIEW 5 of 10 118 Table 2. P450BM3-catalyzed sulfoxidation of thioanisole (3) using H2O2 as the oxidant.a. 119 120 121 Entry DFSM c(4) (µM) ee (%)b F87A variant 1 Im-C6-Phe 232 40 2 Im-C6-Ile 178 38 3 Im-C5-Ile 151 35 4 None 78 6 V78A/F87A variant 5 Im-C6-Phe 103 41 6 Im-C6-Ile 75 40 7 Im-C5-Ile 77 44 8 None 53 4 A74E/F87V/P386S variant 9 Im-C6-Phe 129 47 10 Im-C6-Ile 98 51 11 Im-C5-Ile 108 53 12 None 65 8 a Reaction conditions: P450BM3 (0.5 μM), thioanisole (4 mM), H2O2 122 (20 mM), DFSM (0.5 mM), in phosphate buffer. Reaction time: 30 123 minutes; b Determined by Gas Chromatography. 124 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]. 127 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). 129 When this system was applied (Table 3), reaction rates were significantly decreased (reaction 130 times 18 hours), while at the same time the turnover numbers of the biocatalyst were improved, 131 compared to the use of H2O2 they were five times greater. The low concentration of H2O2 available 132 slowed down both the reaction rate and the oxidative inactivation. We expect that further optimized 133 reaction schemes may provide optimal H2O2 generation rates, ensuring maximized enzymatic 134 sulfoxidation while minimizing the H2O2-related inactivation of the heme enzyme. Again, in the 135 absence of any DFSM, near racemic product was observed, indicating predominant spontaneous 136 sulfoxidation. 137 Table 3. P450BM3 F87A-catalyzed sulfoxidation of 3 using in situ generation of H2O2 by the PpAOx-138 catalyzed oxidation of methanol.a. 139 Entry DFSM c(4) (µM) ee (%) b F87A variant 1Im-C6-Phe 232 40 2Im-C6-Ile 178 38 3Im-C5-Ile 151 35 4None 78 6 V78A/F87A variant 5Im-C6-Phe 103 41 6Im-C6-Ile 75 40 7Im-C5-Ile 77 44 8None 53 4 A74E/F87V/P386S variant 9Im-C6-Phe 129 47 10 Im-C6-Ile 98 51 11 Im-C5-Ile 108 53 12 None 65 8 a Reaction conditions: P450BM3 (0.5 µ M), thioanisole (4 mM), H 2 O 2 (20 mM), DFSM (0.5 mM), in phosphate buffer. Reaction time: 30 min; bDetermined by Gas Chromatography. Table 3. P450BM3 F87A-catalyzed sulfoxidation of 3 using in situ generation of H 2 O 2 by the PpAOx-catalyzed oxidation of methanol.a. Molecules 2019, 24, x FOR PEER REVIEW 6 of 10 140 141 Entry DFSM c(4) (µM) ee (%)b 1 Im-C6-Phe 381 31 2 Im-C6-Ile 359 33 3 Im-C5-Ile 320 30 4 None 248 ≤3 a Reactions were stopped after 18 hours at room temperature. 142 For further reaction details, see Materials and Methods section; b 143 Determined by Gas Chromatography. 144 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. 146 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]. 149 In this respect, the proposed peroxide-driven reaction offers some interesting possibilities for non-150 aqueous reactions using P450 monooxygenases. 151 To test this hypothesis, we evaluated the epoxidation of styrene using precipitated P450BM3 152 F87A suspended in neat styrene as the reaction medium; the stoichiometric oxidant was tBuOOH 153 (Scheme 4). 154 155 Scheme 4. P450BM3-catalyzed epoxidation in neat styrene using tBuOOH as stoichiometric oxidant. 156 Very pleasingly, under these conditions, epoxidation of styrene was observed in the presence of 157 Im-C5-Ile (TONP450BM3 = 178, TON = [Styrene oxide]/[P450BM3]), while in the absence of Im-C5-Ile, 158 100 turnovers were still observed for the biocatalyst. (R)-2 was obtained with optical purities around 159 15% for both reactions. In the absence of the P450BM3 F87A variant, no product formation was 160 observed, even upon prolonged reaction times. To the best of our knowledge, this is the first example 161 of a P450 monooxygenase reaction under neat conditions. 162 3. Materials and Methods 163 Unless otherwise noted, analytical grade solvents and commercially available reagents were 164 used without further purification. 165 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-C6-167 Met exhibited physical and spectral properties in accordance with those reported [17]. 168 GC (Gas Chromatography) analyses were performed on a Shimazdu GC-2010 Plus (Shimazdu, 169 Kyoto, Japan). For the oxidation of styrene (1) to styrene oxide (2), a ChirasilDex CB (Agilent, Santa 170 Clara, CA, USA, 25 m × 0.32 mm × 0.25 µm) column was employed: Carrier gas He, 100 °C hold 12.50 171 Entry DFSM c(4) (µM) ee (%) b 1Im-C6-Phe 381 31 2Im-C6-Ile 359 33 3Im-C5-Ile 320 30 4None 248 ≤3 a Reactions were stopped after 18 h at room temperature. For further reaction details, see Materials and Methods section; bDetermined by Gas Chromatography. 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. Catalysts 2019,9, 567 6 of 9 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). Molecules 2019, 24, x FOR PEER REVIEW 6 of 10 140 141 Entry DFSM c(4) (µM) ee (%)b 1 Im-C6-Phe 381 31 2 Im-C6-Ile 359 33 3 Im-C5-Ile 320 30 4 None 248 ≤3 a Reactions were stopped after 18 hours at room temperature. 142 For further reaction details, see Materials and Methods section; b 143 Determined by Gas Chromatography. 144 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. 146 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]. 149 In this respect, the proposed peroxide-driven reaction offers some interesting possibilities for non-150 aqueous reactions using P450 monooxygenases. 151 To test this hypothesis, we evaluated the epoxidation of styrene using precipitated P450BM3 152 F87A suspended in neat styrene as the reaction medium; the stoichiometric oxidant was tBuOOH 153 (Scheme 4). 154 155 Scheme 4. P450BM3-catalyzed epoxidation in neat styrene using tBuOOH as stoichiometric oxidant. 156 Very pleasingly, under these conditions, epoxidation of styrene was observed in the presence of 157 Im-C5-Ile (TONP450BM3 = 178, TON = [Styrene oxide]/[P450BM3]), while in the absence of Im-C5-Ile, 158 100 turnovers were still observed for the biocatalyst. (R)-2 was obtained with optical purities around 159 15% for both reactions. In the absence of the P450BM3 F87A variant, no product formation was 160 observed, even upon prolonged reaction times. To the best of our knowledge, this is the first example 161 of a P450 monooxygenase reaction under neat conditions. 162 3. Materials and Methods 163 Unless otherwise noted, analytical grade solvents and commercially available reagents were 164 used without further purification. 165 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-C6-167 Met exhibited physical and spectral properties in accordance with those reported [17]. 168 GC (Gas Chromatography) analyses were performed on a Shimazdu GC-2010 Plus (Shimazdu, 169 Kyoto, Japan). For the oxidation of styrene (1) to styrene oxide (2), a ChirasilDex CB (Agilent, Santa 170 Clara, CA, USA, 25 m × 0.32 mm × 0.25 µm) column was employed: Carrier gas He, 100 °C hold 12.50 171 Scheme 4. P450BM3-catalyzed epoxidation in neat styrene using tBuOOH as stoichiometric oxidant. Very pleasingly, under these conditions, epoxidation of styrene was observed in the presence of Im-C5-Ile (TON P450BM3 =178, TON =[Styrene oxide]/[P450BM3]), while in the absence of Im-C5-Ile, 100 turnovers were still observed for the biocatalyst. (R)- 2 was obtained with optical purities around 15% for both reactions. In the absence of the P450BM3 F87A variant, no product formation was observed, even upon prolonged reaction times. To the best of our knowledge, this is the first example of a P450 monooxygenase reaction under neat conditions. 3. Materials and Methods Unless otherwise noted, analytical grade solvents and commercially available reagents were used without further purification. Dual functional small molecules (DFSMs) were synthesized according to the methodology described in the literature [ 17 ]. Compounds Im-C5-Ile, Im-C5-Phe, Im-C6-Ile, Im-C6-Phe and Im-C6-Met exhibited physical and spectral properties in accordance with those reported [17]. GC (Gas Chromatography) analyses were performed on a Shimazdu GC-2010 Plus (Shimazdu, Kyoto, Japan). For the oxidation of styrene ( 1 ) to styrene oxide ( 2 ), a ChirasilDex CB (Agilent, Santa Clara, CA, USA, 25 m × 0.32 mm × 0.25 µ m) column was employed: Carrier gas He, 100 ◦ C hold 12.50 min, 20 ◦ C min −1 to 225 ◦ C, hold 1 min. Retention times: 1 : 3.0 min; (R)- 2 : 7.5 min; (S)- 2 : 7.9 min and dodecane (internal standard): 9.8 min. For the oxidation of thioanisole ( 3 ) to methyl phenyl sulfoxide ( 4 ) a Lipodex E (Agilent, 50 m × 0.25 mm × 0.25 µ m) column was used: Carrier gas He, 130 ◦ C hold 6.0 min, 20 ◦ C min −1 to 200 ◦ C, hold 5.0 min, 25 ◦ C min −1 to 220 ◦ C hold 1.0 min. Retention times: 3: 4.2 min; dodecane (internal standard): 4.9 min; (S)-4: 11.8 min, and (R)-4: 12.4 min. NMR Spectra were recorded ( 1 H NMR 300 MHz; 13 C NMR 75 MHz) with the solvent peak used as the internal reference (7.26 and 77.0 ppm for 1 H and 13 C, respectively) using an Agilent 400 (400 MHz, Santa Clara, CA, USA) P450BM3 F87A and P450BM3 V78A/F87A were produced according to a previously reported protocol [22]. 3.1. Preparation of P450BM3 A74E/F87V/P386S pET28a P450BM3 A74E F87V P386S was expressed in E. coli BL21 (DE3). First, 5 mL Lysogeny Broth (LB) medium supplemented with 30 µ g mL −1 kanamycin was inoculated with single colonies and grown overnight at 37 ◦ C, 180 rpm for preculture. Then 1 L medium (900 mL Terrific Broth (TB) + 100 mL phosphate buffer pH 7.5) supplemented with kanamycin (30 µ g mL −1 ) was inoculated with the 5 mL preculture and incubated at 37 ◦ C, 180 rpm. When OD 600 reached 0.8 (OD 600 : Absorbance at 600 nm), induction was obtained by adding isopropyl β -D-1-thiogalactopyranoside ( 100 µmol L−1 ); at this timepoint 5-aminolevulinic acid (500 µ mol L −1 ) FeSO 4 (100 µ mol L −1 ) were also added. Cultures were then stirred at 30 ◦ C, 180 rpm overnight. Cells were harvested by centrifugation (10,000 g, 30 min, 4 ◦ C) and resuspended in 50 mM phosphate buffer pH 7.5 supplemented with RNase and DNase. After 30 min on ice, cells were disrupted with a French press. The cells debris was removed by centrifugation (14,000 rpm, 30 min, 4 ◦ C). Purification was performed by nickel affinity chromatography using a 60 mL His-Trap FF crude column (GE Healthcare, Chicago, IL, USA) applying a gradient of Catalysts 2019,9, 567 7 of 9 imidazole. The enzyme was then desalted with a PD-10 column and concentrated with an Amicon filter with a cut-offof 30 kDa. 3.2. 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. (S)-2-(4-(1H-imidazol-1-yl)butanamido)-3-phenylpropanoic acid (Im-C4-Phe): Colorless oil (135.4 mg, 45% yield). 1 H-NMR (300 MHz, d 6 -DMSO): δ (ppm) 8.97 (s, 1H), 8.13 (d, 1H, J=8.0 Hz), 7.91 (s, 1H), 7.62 (s, 1H), 7.31–7.14 (m, 5H). 4.37–4.29 (m, 1H), 4.13 (t, 2 H, J=7.5 Hz), 3.07–3.01 (m, 1H), 2.82–2.74 (m, 1H), 2.07-1.96 (m, 4H). 13 C-NMR (75.4 MHz, d 6 -DMSO): δ (ppm) 174.2, 172.6, 138.3, 135.6, 129.2 (2C), 128.7, 126.6 (2C), 124.8, 121.2, 54.1, 48.9, 37.1, 35.2, 25.3. HRMS: m/z calculated for C16H19N3O3(M+): 302.1499; found: 302.1498. (S)-2-(4-(1H-imidazol-1-yl)butanamido)-3-methylpentanoic acid (Im-C4-Ile): Colorless oil (137.7 mg, 49% yield). 1H-NMR (300 MHz, d6-DMSO): δ(ppm) 9.12 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.67 (s, 1H), 4.16–4.11 (m, 1H), 3.92 (t, 2H, J=7.9 Hz), 2.14–2.07 (m, 4H), 1.93-1.87 (m, 2H), 1.21–1.13 (m, 3H), 0.89–0.80 (m, 6H). 13 C-NMR (75.4 MHz, d 6 -DMSO): δ (ppm) 174.1, 171.3, 137.6, 128.6, 119.8, 57.2, 49.0, 36.2, 34.4, 32.1, 31.9, 25.2, 16.1, 11.8. HRMS: m/z calculated for C 14 H 23 N 3 O 3 (M + ): 281.1739; found: 281.1734. 3.3. 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 µ M) 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. 3.4. General Method for the Biocatalyzed Oxidations Employing the H2O2In Situ Generation System P450BM3 F87A (0.5 µ M) 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. Catalysts 2019,9, 567 8 of 9 3.5. 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 µ L) 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. 4. 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. Author Contributions: G.d.G. and F.H. devised the experiments, S.J.-P.W. and F.T. performed the experiments and analyzed the experimental data, M.G. and V.B.U. provided resources and contributed to the experimental design. All authors contributed to the data interpretation and composition of the manuscript. Funding: G.d.G. thanks MINECO (CTQ2016-76908-C2-1,2-P and Ram ó n y Cajal Program) for financial support. F.H. gratefully acknowledges funding by the European Research Commission (ERC consolidator grant, No.648026), the European Union (H2020-BBI-PPP-2015-2-1- 720297), and the Netherlands Organisation for Scientific Research (VICI grant, No. 724.014.003). Acknowledgments: The publication of this article was funded by Delft University of Technology (TU Delft). Conflicts of Interest: The authors declare no conflict of interest. References 1. Urlacher, V.B.; Girhard, M. Cytochrome p450 monooxygenases in biotechnology and synthetic biology. Trends Biotechnol. 2019. 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