Cytochrome P450 Can Epoxidize an Oxepin to a Reactive 2,3-Epoxyoxepin Intermediate: Potential Insights Into Metabolic Ring-Opening of Benzene

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Article Cytochrome P450 Can Epoxidize an Oxepin to a Reactive 2,3-Epoxyoxepin Intermediate: Potential Insights into Metabolic Ring-Opening of Benzene

Holly M. Weaver-Guevara 1,*, Ryan W. Fitzgerald 2, Noah A. Cote 2 and Arthur Greenberg 2,*

1 Department of Chemistry, Grove City College, Grove City, PA 16127, USA 2 Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA; [email protected] (R.W.F.); [email protected] (N.A.C.) * Correspondence: [email protected] (H.M.W.-G.); [email protected] (A.G.); Tel.: +1-603-862-1180 (A.G.) Academic Editor: Edward Lee-Ruﬀ Received: 10 September 2020; Accepted: 1 October 2020; Published: 3 October 2020

Abstract: Dimethyldioxirane epoxidizes 4,5-benzoxepin to form the reactive 2,3-epoxyoxepin intermediate followed by very rapid ring-opening to an o-xylylene that immediately isomerizes to the stable product 1H-2-benzopyran-1-carboxaldehyde. The present study demonstrates that separate incubations of 4,5-benzoxepin with three cytochrome P450 isoforms (2E1, 1A2, and 3A4) as well as pooled human liver microsomes (pHLM) also produce 1H-2-benzopyran-1-carboxaldehyde as the major product, likely via the 2,3-epoxyoxepin. The reaction of 4,5-benzoxepin with cerium (IV) ammonium nitrate (CAN) yields a dimeric oxidized molecule that is also a lesser product of the P450 oxidation of 4,5-benzoxepin. The observation that P450 enzymes epoxidize 4,5-benzoxepin suggests that the 2,3-epoxidation of oxepin is a major pathway for the ring-opening metabolism of benzene to muconaldehyde.

Keywords: 2,3-epoxyoxepins; metabolic ring opening of benzene; cytochrome P450 isozymes

1. Introduction Benzene (1) is a very widespread environmental contaminant and human carcinogen with a complex metabolic fate (Scheme1). The discovery [ 1] of the equilibrium between benzene oxide (2) and oxepin (6) laid the groundwork for the study by Davies and Whitham [2] in which they oxidized oxepins by employing meta-chloroperbenzoic acid (m-CPBA), a study relevant to the metabolism of benzene (1). The oxidation reactions produced acyclic dicarbonyl compounds (e.g., 5 through putative 2,3-epoxyoxepins (7, see Scheme1). However, the reactive 2,3-epoxyoxepin intermediates could neither be isolated nor observed spectroscopically [2], due, in part, to the acidic conditions of their generation. The present study makes a comparison between the oxidative metabolism of benzene through benzene oxide/oxepin with that of a “non-natural metabolite” of naphthalene: 4,5-benzoxepin. It is worthwhile to very brieﬂy compare these metabolic pathways. Benzene is a very widespread environmental contaminant and human carcinogen with a complex metabolic fate (Scheme1) [ 3–6]. It is metabolized in the liver, primarily by cytochrome P450 2E1, mostly through epoxidation to benzene oxide (2)[7–10], followed by rearrangement to phenol (3) and subsequent further oxidation as well as bioconjugation reactions. A relatively small fraction (up to 10–20%) is metabolically ring opened by P450 to Z,Z-muconaldehyde (Z,Z-5) which isomerizes to E,E-5, which is oxidized by aldehyde dehydrogenase to E,E-muconic acid (E,E-4, detected in urine [11]) [12–15]. Muconaldehyde is a very potent inhibitor of the normal generation of blood cells [6]. It reacts with

Molecules 2020, 25, 4542; doi:10.3390/molecules25194542 www.mdpi.com/journal/molecules

Article Cytochrome P450 Can Epoxidize an Oxepin to a Reactive 2,3-Epoxyoxepin Intermediate: Potential Insights into Metabolic Ring-Opening of Benzene

Holly M. Weaver-Guevara 1,*, Ryan W. Fitzgerald 2, Noah A. Cote 2 and Arthur Greenberg 2,*

Academic Editor: Edward Lee-Ruff Received: 10 September 2020; Accepted: 1 October 2020; Published: 3 October 2020

Keywords: 2,3-epoxyoxepins; Metabolic Ring Opening of Benzene; Cytochrome P450 Isozymes

Scheme 1. Metabolic pathway of benzene to phenol and postulated pathway to E,E-muconic acid. Molecules 2020, 25, x; doi: www.mdpi.com/journal/molecules Benzene is unique in its conversion to significant quantities of closed-ring (e.g., phenol, 3) and ring-opened (e.g., muconic acid, 4) metabolites via mammalian monooxygenases. Alkylated aromatics such as toluene, ethylbenzene, and xylenes are predominantly metabolically oxidized at the alkyl groups (e.g., toluene to benzyl alcohol [19]). Naphthalene is metabolized to 1-naphthol. Other, larger polycyclic aromatic hydrocarbons (PAH) also form ring-substituted rather than open-chain metabolites. While there is near-universal acceptance of the benzene oxide pathway to phenol, the pathway to muconaldehyde is less understood. Direct attack by hydroxyl (OH) on benzene [20] followed by a reaction of the initial radical with molecular oxygen appears to be relevant to atmospheric formation of muconaldehyde [21]. Of particular interest is the pathway suggested by Davies and Whitham [2] nearly four decades ago. They postulated the epoxidation of oxepin to “2,3-epoxyoxepin” (7) followed by rapid ring opening to Z,Z-muconaldehyde (Z,Z-5). Z,Z-5 isomerizes thermally as well as catalytically to E,Z-5 followed by catalyzed isomerization to E,E-5 (see Scheme1) [22,23]. Using the acidic epoxidation reagents (e.g., m-CPBA) of the period, Davies and Whitham implicated the intermediacies of 2,3-epoxyoxepins without isolating or spectroscopically observing them [2]. Ab initio calculations predicted a low barrier for the concerted ring opening of 7 to Z,Z-5 [24]. Interestingly, 3,6-bridged 2,3-epoxyoxepins were reported in 1982, synthesized using m-CPBA and exhibited melting points ca 100 ◦C[25]. These surprising stabilities are attributable to the influences of the bridges which both frustrate thermal rearrangements and somewhat stabilize the epoxides to acid [26]. The first report of a simple 2,3-epoxyoxepin involved reaction of 2,7-dimethyloxepin (8) with dimethyldioxirane (DMDO) [also methyl(trifluoromethyl)dioxirane] at 60 C involved NMR − ◦ observation of a trace amount of 2,3-epoxyoxepin 9 which isomerized to the diketone (Z,Z-10) at 20 C (Scheme2A) [ 27]. A similar study of the parent 2/6 equilibrium produced Z,Z-10 but no − ◦ direct observation of 7 [28]. The reaction of DMDO with 4,5-benzoxepin (11) at 50 C provided a − ◦ near quantitative yield of 12 which rearranged rapidly at 15 C to form 14 almost quantitatively, − ◦ undoubtedly via the short-lived ortho-xylylene 13 (Scheme2B) [29]. Molecules 2020, 25, x 2 of 12

Scheme 1. Metabolic pathway of benzene to phenol and postulated pathway to E,E-muconic acid.

Benzene is a very widespread environmental contaminant and human carcinogen with a complex metabolic fate (Scheme 1) [3–6]. It is metabolized in the liver, primarily by cytochrome P450 2E1, mostly through epoxidation to benzene oxide (2) [7–10], followed by rearrangement to phenol (3) and subsequent further oxidation as well as bioconjugation reactions. A relatively small fraction (up to 10–20%) is metabolically ring opened by P450 to Z,Z-muconaldehyde (Z,Z-5) which isomerizes to E,E-5, which is oxidized by aldehyde dehydrogenase to E,E-muconic acid (E,E-4, detected in urine [11]) [12–15]. Muconaldehyde is a very potent inhibitor of the normal generation of blood cells [6]. It reacts with glutathione and can cross-link DNA and proteins and induces inhibition of gap junction intracellular communication [16–18]. Benzene is unique in its conversion to significant quantities of closed-ring (e.g., phenol, 3) and ring-opened (e.g., muconic acid, 4) metabolites via mammalian monooxygenases. Alkylated aromatics such as toluene, ethylbenzene, and xylenes are predominantly metabolically oxidized at the alkyl groups (e.g., toluene to benzyl alcohol [19]). Naphthalene is metabolized to 1-naphthol. Other, larger polycyclic aromatic hydrocarbons (PAH) also form ring-substituted rather than open- chain metabolites. While there is near-universal acceptance of the benzene oxide pathway to phenol, the pathway to muconaldehyde is less understood. Direct attack by hydroxyl (OH) on benzene [20] followed by a reaction of the initial radical with molecular oxygen appears to be relevant to atmospheric formation of muconaldehyde [21]. Of particular interest is the pathway suggested by Davies and Whitham [2] nearly four decades ago. They postulated the epoxidation of oxepin to “2,3- epoxyoxepin” (7) followed by rapid ring opening to Z,Z-muconaldehyde (Z,Z-5). Z,Z-5 isomerizes thermally as well as catalytically to E,Z-5 followed by catalyzed isomerization to E,E-5 (see Scheme 1) [22,23]. Using the acidic epoxidation reagents (e.g., m-CPBA) of the period, Davies and Whitham implicated the intermediacies of 2,3-epoxyoxepins without isolating or spectroscopically observing them [2]. Ab initio calculations predicted a low barrier for the concerted ring opening of 7 to Z,Z-5 [24]. Interestingly, 3,6-bridged 2,3-epoxyoxepins were reported in 1982, synthesized using m-CPBA and exhibited melting points ca 100 °C [25]. These surprising stabilities are attributable to the influences of the bridges which both frustrate thermal rearrangements and somewhat stabilize the epoxides to acid [26]. The first report of a simple 2,3-epoxyoxepin involved reaction of 2,7-dimethyloxepin (8) with dimethyldioxirane (DMDO) [also methyl(trifluoromethyl)dioxirane] at −60 °C involved NMR observation of a trace amount of 2,3-epoxyoxepin 9 which isomerized to the diketone (Z,Z-10) at −20 °C (Scheme 2A) [27]. A similar study of the parent 2/6 equilibrium produced Z,Z-10 but no direct observation of 7 [28]. The reaction of DMDO with 4,5-benzoxepin (11) at −50 °C provided a near quantitativeMolecules yield2020, 25 of, 4542 12 which rearranged rapidly at −15 °C to form 14 almost quantitatively,3 of 12 undoubtedly via the short-lived ortho-xylylene 13 (Scheme 2B) [29].

Scheme 2. (A) Reaction of 2,7-dimethyloxepin with dimethyldioxirane (DMDO); (B) reaction of Scheme 4,5-benzoxepin2. (A) Reaction with of dimethyldioxirane. 2,7-dimethyloxepin with dimethyldioxirane (DMDO); (B)reaction of 4,5- benzoxepin with dimethyldioxirane. The growing acceptance that oxepin is an intermediate in the pathway to muconaldehyde has raised Moleculesother 20 subtleties.20, 25, x Low-temperature DMDO reactions with oxepin, 2-methyloxepin, and 2,7-dimethyloxepin3 of 12 Theform growing the Z,Z -isomersacceptance (e.g., Zthat,Z-5, oxepin Scheme3 )is [ 28an]. Viewingintermediate consecutive in the one-electron pathway oxidations, to muconaldehyde as another has raisedreactions otherreasonable subtleties.[30]. pathway The reaction Low to muconaldehyde,-temperature of the above the three DMDO Golding oxepins group reactions exploredwith cerium(IV) with surrogate oxepin, reactions ammonium 2-methyloxepin, [30]. The nitrate reaction (CAN) and 2,7- dimethyloxepinproducedof the aboveE,Z form-isomers three the oxepins Z exclusively,Z- withisomers cerium(IV) (e.g., (e.g., E ammonium ,ZZ,-Z5 -5as, Scheme well nitrate as (CAN)3)E, Z[28].-10 produced, Viewing see SchemeE,Z -isomersconsecutive 3), consistent exclusively one - withelectron oxidations,geometric(e.g., asE , Zisomerizationanother-5 as well reasonable as E, Zof-10 the, see pathwayring Scheme-opened3), to consistent muconaldehyde,radical withintermediate geometric the isomerization[30]. Golding Without group of theintercession ring-openedexplored of surrogate free radicalradical intermediates intermediate, [Z30,].Z- Without5 is converted intercession to ofE, freeZ-5 radicalat 55 ° intermediates,C in the darkZ, Zin-5 16is converted h [22]. The to E ,cyclicZ-5 at 2H-

pyran55-2◦C-carboxaldehyde in the dark in 16 his [ 22the]. Thehigher cyclic-energy 2H-pyran-2-carboxaldehyde intermediate connecting is the these higher-energy two stereoisomers. intermediate This thermalconnecting pathway these does two stereoisomers.not isomerize This E,Z thermal-5 to E pathway,E-5. E,Z does-5 (as not well isomerize as Z,EZ,Z-5-)5 areto E ,readilyE-5. E,Z- 5converted(as well to Z Z 5 E E 5 E,E-5as in ,the- )presence are readily of converted a nucleophilic to , - reagentin the presence (e.g., trimethylamine of a nucleophilic in reagent acetonitrile) (e.g., trimethylamine acting as a catalyst in acetonitrile) acting as a catalyst in reversible Michael additions [22]. In contrast, for the 4,5-benzo-substituted in reversible Michael additions [22]. In contrast, for the 4,5-benzo-substituted system (Scheme 2), 1H- system (Scheme2), 1 H-2-benzopyran-1-carboxaldehyde (14), the cyclic molecule is more stable than the 2-benzopyranZ,Z-isomer- (113-carboxal) and effectivelydehyde shuts (14), down the cyclic the pathway molecule to the is moreE,Z-isomer, stable and than is alsothe higherZ,Z-isomer in energy. (13) and effectively shuts down the pathway to the E,Z-isomer, and is also higher in energy.

Scheme 3. Golding dimethyldioxirane (DMDO) and postulated cerium (IV) ammonium nitrate (CAN) Scheme 3. Golding dimethyldioxirane (DMDO) and postulated cerium (IV) ammonium nitrate oxidation mechanisms for oxepin [30]. (CAN) oxidation mechanisms for oxepin [30]. 2,7-Dimethyloxepin (8, Scheme2) is an interesting substrate for investigation of P450 metabolism. Although2,7-Dimethyloxepin it is the only detectable (8, Scheme tautomer, 2) is due an to interesting steric repulsion substrate between for the methylinvestigation substituents of P450 metabolism.in the benzene Although oxide it tautomer, is the only the detectable Curtin–Hammett tautomer, Principle due to does steric not re eliminatepulsion thebetween undetectable the methyl substituentstautomer in from the being benzene the reactive oxide tautomer, substrate [ 31the]. InCurtin principle,–Hammett it could Principle be derived does by P450-catalyzed not eliminate the undetectableepoxidation tautomer of ortho-xylene. from being Since xylenesthe reactive are oxidized substrate at the [31]. methyl In substituent,principle, it8 shouldcould notbe formderived in by P450signiﬁcant-catalyzed quantity epoxidation via mammalian of ortho-xylene. P450 monooxygenases. Since xylenes are One oxidized might think at the of it methyl as a “non-natural substituent, 8 shouldmetabolite not form further in significant down a hypothetical quantity via pathway mammalian avoiding P450 methyl monooxygenases. oxidation”. 4,5-Benzoxepin One might (think11) is of it even more interesting as a non-natural metabolite. Naturally occurring P450-catalyzed epoxidation at as a “non-natural metabolite further down a hypothetical pathway avoiding methyl oxidation”. 4,5- the 1,2-position of naphthalene (15) forms the 1,2-epoxide (16) which isomerizes to 1-naphthol (17) Benzoxepin (11) is even more interesting as a non-natural metabolite. Naturally occurring P450- rather than to the less stable oxepin 18, which lacks aromatic stabilization [32,33]. Hypothetical catalyzedepoxidation epoxidation at the 2,3-position at the 1,2 of- naphthaleneposition of would naphthalene eliminate ( aromaticity—the15) forms the 2,3-epoxide 1,2-epoxide (19 ()16 is) far which isomerizesless stable to than 1-naphthol the 1,2-epoxide. (17) rather It is never than formed, to the and less therefore stable its oxepin valence 18 isomer, which 4,5-benzoxepin lacks aromatic stabilization [32,33]. Hypothetical epoxidation at the 2,3-position of naphthalene would eliminate aromaticity—the 2,3-epoxide (19) is far less stable than the 1,2-epoxide. It is never formed, and therefore its valence isomer 4,5-benzoxepin (11) is not produced naturally (see Scheme 4) [32,33]. Thus, 11 can also be considered as a “non-natural metabolite further down a hypothetical pathway avoiding natural 1,2-epoxidation of naphthalene”. Although the benzene oxide isomer of 8 is undetectable by NMR, it is only a few kcal/mol higher in energy, so that rearrangement to dimethylphenol and attack by nucleophiles should be possible. In contrast, 19 is calculated to be over 30 kcal/mol less stable than 11 [33], eliminating reactions via the epoxide.

Molecules 2020, 25, x 3 of 12 reactions [30]. The reaction of the above three oxepins with cerium(IV) ammonium nitrate (CAN) produced E,Z-isomers exclusively (e.g., E,Z-5 as well as E,Z-10, see Scheme 3), consistent with geometric isomerization of the ring-opened radical intermediate [30]. Without intercession of free radical intermediates, Z,Z-5 is converted to E,Z-5 at 55 °C in the dark in 16 h [22]. The cyclic 2H- pyran-2-carboxaldehyde is the higher-energy intermediate connecting these two stereoisomers. This thermal pathway does not isomerize E,Z-5 to E,E-5. E,Z-5 (as well as Z,Z-5) are readily converted to E,E-5 in the presence of a nucleophilic reagent (e.g., trimethylamine in acetonitrile) acting as a catalyst in reversible Michael additions [22]. In contrast, for the 4,5-benzo-substituted system (Scheme 2), 1H- 2-benzopyran-1-carboxaldehyde (14), the cyclic molecule is more stable than the Z,Z-isomer (13) and effectively shuts down the pathway to the E,Z-isomer, and is also higher in energy.

Scheme 3. Golding dimethyldioxirane (DMDO) and postulated cerium (IV) ammonium nitrate (CAN) oxidation mechanisms for oxepin [30].

2,7-Dimethyloxepin (8, Scheme 2) is an interesting substrate for investigation of P450 metabolism. Although it is the only detectable tautomer, due to steric repulsion between the methyl substituents in the benzene oxide tautomer, the Curtin–Hammett Principle does not eliminate the undetectable tautomer from being the reactive substrate [31]. In principle, it could be derived by P450-catalyzed epoxidation of ortho-xylene. Since xylenes are oxidized at the methyl substituent, 8 should not form in significant quantity via mammalian P450 monooxygenases. One might think of it as a “non-natural metabolite further down a hypothetical pathway avoiding methyl oxidation”. 4,5- Benzoxepin (11) is even more interesting as a non-natural metabolite. Naturally occurring P450- catalyzed epoxidation at the 1,2-position of naphthalene (15) forms the 1,2-epoxide (16) which isomerizes to 1-naphthol (17) rather than to the less stable oxepin 18, which lacks aromatic stabilization [32,33]. Hypothetical epoxidation at the 2,3-position of naphthalene would eliminate Molecules 2020, 25, 4542 4 of 12 aromaticity—the 2,3-epoxide (19) is far less stable than the 1,2-epoxide. It is never formed, and therefore its valence isomer 4,5-benzoxepin (11) is not produced naturally (see Scheme 4) [32,33]. Thus,(11 11) is can not also produced be considered naturally (see as Schemea “non4-natural)[ 32,33]. metabolite Thus, 11 can further also be down considered a hypothetical as a “non-natural pathway avoidingmetabolite natural further 1,2-epoxidation down a hypothetical of naphthalene” pathway avoiding. natural 1,2-epoxidation of naphthalene”. Although the benzene oxide isomer of 8 is undetectable by NMR, it is only a few kcal/mol higher Although the benzene oxide isomer of 8 is undetectable by NMR, it is only a few kcal/mol higher in energy, so that rearrangement to dimethylphenol and attack by nucleophiles should be possible. in energy, so that rearrangement to dimethylphenol and attack by nucleophiles should be possible. In contrast, 19 is calculated to be over 30 kcal/mol less stable than 11 [33], eliminating reactions via In contrast,the epoxide. 19 is calculated to be over 30 kcal/mol less stable than 11 [33], eliminating reactions via the epoxide.

Molecules 2020, 25, x 4 of 12

Scheme 4. Comparison of Epoxidation Pathways for Naphthalene.

2. Results and DiscussionScheme 4. Comparison of Epoxidation Pathways for Naphthalene. 2. Results and Discussion 2.1. Oxidations of 4,5-Benzoxepin with DMDO and CAN 2.1. Oxidations of 4,5-Benzoxepin with DMDO and CAN The present study compares the reaction products of DMDO and CAN oxidations of 2,7- dimethyloxepinThe present (8) study and compares4,5-benzoxepin the reaction (11 products) with ofthose DMDO derived and CAN from oxidations incubation of 2,7-dimethyloxepin with cytochrome P450( 8) enzymes and 4,5-benzoxepin (synthetic ( procedures11) with those found derived in fromAppendix incubation A and with Supplementary cytochrome P450 Information). enzymes (synthetic procedures found in AppendixA and Supplementary Information). Whereas oxidation of Whereas oxidation of 2,7-dimethyloxepin with DMDO produces Z,Z-10 and CAN oxidation yields 2,7-dimethyloxepin with DMDO produces Z,Z-10 and CAN oxidation yields E,Z-10 [29], the corresponding E,Z-10 [29], the corresponding oxidations of 4,5-benzoxepin produced very different, non-isomeric oxidations of 4,5-benzoxepin produced very different, non-isomeric products. A reaction with DMDO products.(1:1 molar A reaction ratio) produces with DMDO14 but reaction (1:1 molar with CANratio) (2:1 produces molar ratio) 14 but produces reaction two with essentially CAN dimeric (2:1 molar ratio) produces two essentially dimeric molecules assigned structures 21 (C20H16O4) and 20 (C20H14O4) molecules assigned structures 21 (C20H16O4) and 20 (C20H14O4) (Scheme5). The two compounds appear (Schemeto form 5). viaThe different two compounds pathways. appear As will beto supportedform via different later in this pathways. paper, incubation As will ofbe11 supportedwith P450 later in thisisoforms paper, yielded incubatio20 asn a lesser of 11product with P450 with aisoforms minute quantity yielded of 2021. as a lesser product with a minute quantity of 21.

O O O H O O H O O O 20 21 SchemeScheme 5. Proposed 5. Proposed products products of of the the CAN CAN reaction withwith 4,5-benzoxepin 4,5-benzoxepin (11 ()11 assigned) assigned structure structure21 21 (C20H16O4) and 20 (C20H14O4). (C20H16O4) and 20 (C20H14O4). Spectroscopic data in support of the identifications of 20 and 21 are in the Experimental Section andSpectroscopic further documented data in support in the Supplementary of the identifications Information. of B3LYP20 and/6-31G* 21 are calculations in the Experimental [34,35] predict Section and that further (R,R )- documented (or S,S)-20 is 1.4 in kcal the/mol Supplementary lower in energy Information. than meso-20. The B3LYP/6 anti- (C-31G*i) diastereomer calculations of 21 [34,35]is 13 predictcalculated that (R, toR) be- (or 5.7 S kcal,S)-/20mol is lower1.4 kcal/mol in energy lower than in the energy syn- (C tha2v)n diastereomer. meso-20. The antiC NMR- (Ci) shows diastereomer ten 1 of 21unique is calculated carbon to atoms be 5.7 with kcal/mol shifts consistent lower in with energy those than expected the syn for- 20(C.2v)H diastereomer. NMR shows seven 13C NMR distinct shows ten uniqueprotons (4H:carbon aromatic; atoms 2H with vinylic; shifts 1H consistent at 6.6 ppm roughlywith those consistent expected with thefor downfield 20. 1H NMR shift calculatedshows seven distinct protons (4H: aromatic; 2H vinylic; 1H at 6.6 ppm roughly consistent with the downfield shift calculated for the dimer (meso or R,R/S,S)). Two-dimensional NMR (COSY, DEPT, NOESY, and HSQC) suggests the presence of a conjugated alkene (NOESY correlation) and confirms the presence of one proton (6.6 ppm) on the carbon alpha to the carbonyl (DEPT, HSQC correlations). IR spectroscopy indicates a strong absorbance at 1666 cm-1 and no absorbance due to hydroxyl groups. The UV absorbances (λmax = 273 nm and 338 nm) are also consistent with similar structural features. GC/EI-MS data as well as ESI/MS data were not definitive for a structure corresponding to 21 (expected m/z = 318, or 319 via ESI/MS). However, EI and ESI/MS are both consistent with isocoumarin which could arise from 20 through a loss of acetylene (or its equivalent, see Scheme 6A), presumably in a stepwise mechanism. Column chromatography did not provide samples of 20 or 21 suitable for elemental analysis or X-ray crystallographic study. The EI mass spectrum observed corresponding to isocoumarin (22) includes an abundant m/z 90 (Parent - 2 CO) in agreement with a published mass spectrum [36]. Another published mass spectrum for isocoumarin [37] appears to erroneously report an abundant m/z 97 with no m/z 90 reported. Precise mass analysis corresponds to the formula C9H6O2 for the m/z 146 ion as expected for isocoumarin (see Supplementary Information). The ESI/MS data also correspond to isocoumarin. Further investigation of this system involved synthesis of the reported monomeric bromo derivative 23 [38]. Coupling using NiCl2 and Zn in the presence of pyridine and 2,2′-bipyridine to produce dimer 24 was attempted. It provided lactone 25 (Scheme 6B) in addition to reduced monomer 26, the latter likely due to residual moisture. Although the expulsion of acetylene appears to be unlikely, it is possible that the two carbons lost en route to 25 might arise in other forms under these reaction conditions. A single small-scale headspace analysis found no acetylene and only methane as the

Molecules 2020, 25, 4542 5 of 12

for the dimer (meso or R,R/S,S)). Two-dimensional NMR (COSY, DEPT, NOESY, and HSQC) suggests the presence of a conjugated alkene (NOESY correlation) and confirms the presence of one proton (6.6 ppm) on the carbon alpha to the carbonyl (DEPT, HSQC correlations). IR spectroscopy indicates 1 a strong absorbance at 1666 cm− and no absorbance due to hydroxyl groups. The UV absorbances (λmax = 273 nm and 338 nm) are also consistent with similar structural features. GC/EI-MS data as well as ESI/MS data were not definitive for a structure corresponding to 21 (expected m/z = 318, or 319 via ESI/MS). However, EI and ESI/MS are both consistent with isocoumarin which could arise from 20 through a loss of acetylene (or its equivalent, see Scheme6A), presumably in a stepwise mechanism. Column chromatography did not provide samples of 20 or 21 suitable for elemental analysis or X-ray crystallographic study. The EI mass spectrum observed corresponding to isocoumarin (22) includes an abundant m/z 90 (Parent - 2 CO) in agreement with a published mass spectrum [36]. Another published mass spectrum for isocoumarin [37] appears to erroneously report an abundant m/z 97 with no m/z 90 reported. Precise mass analysis corresponds to the formula C9H6O2 for the m/z 146 ion as expected for isocoumarin (see Supplementary Information). The ESI/MS data also correspond to isocoumarin. Further investigation of this system involved synthesis of the reported monomeric bromo derivative 23 [38]. Coupling using NiCl2 and Zn in the presence of pyridine and 2,20-bipyridine to produce dimer 24 was attempted. It provided lactone 25 (Scheme6B) in addition to reduced monomer 26, the latter likely due to residual Molecules 20moisture.20, 25, x Although the expulsion of acetylene appears to be unlikely, it is possible that the two carbons 5 of 12 lost en route to 25 might arise in other forms under these reaction conditions. A single small-scale single gaseousheadspace component analysis found, no which acetylene was and very only methane surprising as the single and gaseousis worthy component, of future which was systematic investigation.very surprising and is worthy of future systematic investigation.

O O O EI MS O CH A) 2 + HC O or O ? ESI MS 20 22

NiCl2, Zn dust pyridine O 2,2’-bipyridine O O acetonitrile B) 2 Br O O O 23 24?

O O 2 + CH + O HC ? O 25 26 Scheme 6. (A) Observed mass spectrometry fragmentation of 20 to 22;(B) Postulated pathway for Scheme observed6. (A) Observed chemical transformation mass spectrometry of 23 to 26 fragmentation. of 20 to 22; (B) Postulated pathway for observed chemical transformation of 23 to 26. Scheme7 describes a postulated reaction mechanism for the formation of the dimeric 20 from 4,5-benzoxepin under CAN oxidation. While the mechanism resembles that postulated by Scheme 7 describes a postulated reaction mechanism for the formation of the dimeric 20 from Golding et al. [30] in its initial steps, the ring opening of the postulated free radical 25 is unfavorable 4,5-benzoxepindue to the under need to CAN lose aromaticity; oxidation. hence, While14 (viathe13 mechanism) is not formed resembles and isomerization that postulated to E,Z-13 should by Golding et al. [30]not in occur. its initial While steps, the mechanism the ring o ﬀopeningered in Scheme of the7 depictspostulated dimerization free radical through 25 the is reaction unfavorable of the due to the needradical to lose24 aromaticitywith 11, one would; hence expect, 14 analogous(via 13) is dimerization not formed through and isomerization25 to be more sterically to E,Z hindered.-13 should not occur. WhileThe central the mechanism C–C linkage inoffered dimeric in molecule Scheme20 7might depicts be anticipated dimerization to be somewhatthrough the weak reaction due to of the the possibility of forming two capto-dative free radicals. However, a dimeric molecule capable of radical 24 with 11, one would expect analogous dimerization through 25 to be more sterically hindered. The central C–C linkage in dimeric molecule 20 might be anticipated to be somewhat weak due to the possibility of forming two capto-dative free radicals. However, a dimeric molecule capable of forming two PhCOC(OPh)(Ph) radicals showed no evidence of decomposition at 60 °C [39]. Indeed, a crystallographic study of bridged dimer containing this linkage provided a C–C bond length of 1.523 Ǻ [40]. Other stable examples are known [41,42]. The pathway to formation of 21 is suggested by the oxidation of the bridged oxepin to form 30, presumed to occur via glycol 31 [43]. The equivalent 32 and 33 are reasonable precursors to 21 (Scheme 8).

Molecules 2020, 25, x 5 of 12

single gaseous component, which was very surprising and is worthy of future systematic investigation.

O O O EI MS O CH A) 2 + HC O or O ? ESI MS 20 22

NiCl2, Zn dust pyridine O 2,2’-bipyridine O O acetonitrile B) 2 Br O O O 23 24?

O O 2 + CH + O HC ? O 25 26

Scheme 6. (A) Observed mass spectrometry fragmentation of 20 to 22; (B) Postulated pathway for observed chemical transformation of 23 to 26.

Scheme 7 describes a postulated reaction mechanism for the formation of the dimeric 20 from 4,5-benzoxepin under CAN oxidation. While the mechanism resembles that postulated by Golding et al. [30] in its initial steps, the ring opening of the postulated free radical 25 is unfavorable due to the need to lose aromaticity; hence, 14 (via 13) is not formed and isomerization to E,Z-13 should not occur. While the mechanism offered in Scheme 7 depicts dimerization through the reaction of the radical 24 with 11, one would expect analogous dimerization through 25 to be more sterically Molecules 2020, 25, 4542 6 of 12 hindered. The central C–C linkage in dimeric molecule 20 might be anticipated to be somewhat weak due to the possibility of forming two capto-dative free radicals. However, a dimeric molecule capable crystallographic study of bridgedof dimer formingforming containing two two this PhCOC(OPh)(Ph) PhCOC(OPh)(Ph) li radicals radicals showed showed no evidence no evidence of decomposition of decomposition at 60 ◦C[39 at]. Indeed,60 °C [39]. Indeed,a crystallographic a crystallographic study ofstudy bridged of bridged dimer containing dimer containing this linkage this provided linkage a C–C provid bonded length a C–C of bond nkage provided a C–C bond length of 1.5231.523 Ǻ [40].[40]. Other Other stable examples are known [41,42]. The pathway to formation of 21 is suggested by length of 1.523 Ǻ [40]. Other stable examples are known [41,42]. The pathway to formation of 21 is the oxidation of the bridged oxepin to form 30, presumed to occur via glycol 31 [43]. The equivalent 32 suggested by the oxidation of the bridged oxepin to form 30, presumed to occur via glycol 31 [43]. and 33 are reasonable precursors to 21 (Scheme8). The equivalent 32 and 33 are reasonable precursors to 21 (Scheme 8).

Molecules 2020, 25, x 6 of 12

Scheme 7. Postulated mechanism for formation of the dimeric molecule 20 from 4,5-benzoxepin in Scheme 7. Postulated mechanism for formation of the dimeric molecule 20 from 4,5-benzoxepin in the the presence of CAN. presence of CAN.

Scheme 8. Models (30 and 31) for and potential precursors (32 and 33) of 21. Scheme 8. Models (30 and 31) for and potential precursors (32 and 33) of 21. 2.2. Incubations of 4,5-Benzoxepin with P450 Isoforms 2.2. IncubationsIncubations of 4,5- ofBenzoxepin11 with P450 with 2E1,P450 P450 Isoforms 1A2, P450 3A4, and pooled human liver microsomes Incubations(pHLM) were of compared 11 with with P450 the 2E1, products P450 of 1A2, DMDO P450 and 3A4, CAN andoxidations. pooled The human 2E1 isoform liver is microsomes known to be optimal for benzene; the 1A2 isoform is optimal for naphthalene; the 3A4 isoform has a larger (pHLM) were compared with the products of DMDO and CAN oxidations. The 2E1 isoform is known active site and is relatively non-speciﬁc. The enzymes and pHLM were obtained commercially and to be optimal for benzene; the 1A2 isoform is optimal for naphthalene; the 3A4 isoform has a larger the co-factor mixture is described in the Experimental Section. The primary analytical method for the activeenzyme site and incubation is relatively products non was-specific. GC/MS The since enzymes the scale and is small. pHLM were obtained commercially and the co-factorFigure mixture1A reproduces is described the GC in /theMS Experiment chromatogramal Section. derived fromThe theprimary incubation analytical of 4,5-benzoxepin method for the enzyme(11) incubation with cytochrome products P450 1A2.was HighlightedGC/MS since in this the chromatogram scale is small. are unreacted 11, 1H-2-benzopyran-1 -carboxaldehydeFigure 1A reproduces (14) and the a smaller GC/MS quantity chromatogram of the dimer derived (20). Figure from1B the reproduces incubation the massof 4,5 spectra-benzoxepin of (11) these with three cytochrome components. P450 We 1A2. have establishedHighlighted that inthe epoxidationthis chromatogram of 11 with DMDO are unreacted cleanly produces 11, 1H-2- benzopyran-1-carboxaldehyde (14) and a smaller quantity of the dimer (20). Figure 1B reproduces the mass spectra of1 these three components. We have established that the epoxidation of 11 with

DMDO cleanly produces 14 [29]. Trials with P450 2E1, P450 3A4 and pHLM all produced similar results. Incubations of 11 with 2E1 and 3A4 yielded a lower proportion of 21 relative to 14. The reaction of 11 with CAN produces not 14 but the oxidized dimeric molecule 21, as noted above. Control experiments without the P450 isoforms did not yield 14 or 20. Incubations with 2E1, 3A4, and pHLM each yield very similar results with 4,5-benzoxepin. They suggest that, for the cytochrome P450s tested, each epoxidize the oxepin structure as the major pathway. It may well be that consecutive single-electron oxidations constitute a relatively minor pathway.

2.3. Incubations of 2,7-Dimethyloxepin with P450 Isoforms Incubations of 2,7-dimethyloxepin (8) with the same suite of enzymes as 11 under the same reaction conditions resulted in inconsistent observations of E,Z-10 in very trace amounts. Some of the trials provided dimethylphenols in appreciable yields and dimethyldihydroxybenzenes in low-to- moderate yields. Control experiments suggest that these products are the result of enzymatic reactions, not just thermal rearrangements. There has been no evidence of oxidation at the methyl groups of 8 to form carboxylic acid derivatives. The paper by Golding et al. [30] did not report P450 incubations. As noted earlier, the benzene oxide valence isomer of 2,7-dimethyloxepin is only a few kcal/mol higher in energy than the benzene oxide isomer. Benzene oxides exhibit very short half-lives in protic solvents consistent with the observation of dimethylphenol(s) and even dimethylcatechol(s). In contrast, the epoxide valence isomer (19) of 4,5-benzoxepin is energetically inaccessible, shutting down some pathways of competing reactions, thus permitting epoxidation of 11 and its rearrangement to 14. The GC/MS data for the incubations of 8 are included in the Supplementary Information.

Molecules 2020, 25, 4542 7 of 12

14 [29]. Trials with P450 2E1, P450 3A4 and pHLM all produced similar results. Incubations of 11 with 2E1 and 3A4 yielded a lower proportion of 21 relative to 14. The reaction of 11 with CAN produces not 14 but the oxidized dimeric molecule 21, as noted above. Control experiments without the P450 isoforms did not yield 14 or 20. Incubations with 2E1, 3A4, and pHLM each yield very similar results with 4,5-benzoxepin. They suggest that, for the cytochrome P450s tested, each epoxidize the oxepin structure as the major pathway. It may well be that consecutive single-electron oxidations constitute a relatively minor pathway.

2.3. Incubations of 2,7-Dimethyloxepin with P450 Isoforms Incubations of 2,7-dimethyloxepin (8) with the same suite of enzymes as 11 under the same reaction conditions resulted in inconsistent observations of E,Z-10 in very trace amounts. Some of the trials provided dimethylphenols in appreciable yields and dimethyldihydroxybenzenes in low-to-moderate yields. Control experiments suggest that these products are the result of enzymatic reactions, not just thermal rearrangements. There has been no evidence of oxidation at the methyl groups of 8 to form carboxylic acid derivatives. The paper by Golding et al. [30] did not report P450 incubations. As noted earlier,Molecules the 20 benzene20, 25, x oxide valence isomer of 2,7-dimethyloxepin is only a few kcal/mol higher in7 energy of 12 than the benzene oxide isomer. Benzene oxides exhibit very short half-lives in protic solvents consistent with the observation of dimethylphenol(s) and even dimethylcatechol(s). In contrast, the epoxide valence isomer (19) of 4,5-benzoxepin is energetically inaccessible, shutting down some pathways of competing reactions, thus permitting epoxidation of 11 and its rearrangement to 14. The GC/MS data for the incubations of 8 are included in the Supplementary Information.

5.6 6.1 6.6 7.1 7.6 8.1 8.6 9.1 9.6 10.1 10.6 11.1 11.6 12.1 12.6 13.1 13.6

(A)

100 13 1 1 100 115 100 80 80 80 9 144 60 60 146 60

40 40 40 7 1 6

20 20 20 63 8 51 160

0 0 0

36 44 53 61 69 77 86 96 36 46 58 67 79 90

35 43 53 73 82 91

105 115 123 132 146 166 193 102 116 127 138 149 161 177 192

110 120 133 141 154 162 175 190 101 (B)

FigureFigure 1. ( 1.A )(A Total) Total Ion Ion Chromatogram Chromatogram (TIC) (TIC) for for reaction reaction of of 4,5-benzoxepin4,5-benzoxepin 11 withwith P450 P450 1A2; 1A2; (B (B) m) massass spectraspectra of compoundsof compounds11 ,1114, 14, and, and20 20, respectively., respectively.

Molecules 2020, 25, 4542 8 of 12

3. Materials and Methods Starting materials, reagents, and solvents were purchased from Aldrich, Alfa Aesar and Fisher Scientific and used without further purification unless otherwise indicated. Reaction products were purified by flash column chromatography using Silicycle Inc. silica gel (60 Å, 230–400 mesh). Thin-layer chromatography was performed on Agela Technologies TLC Silica plates (Agela Technologies, Tianjin, China) (silica gel 60 GF254) and visualization was accomplished with UV light (UVP, INC, San Gabriel, CA, USA). 2,7-dimethyloxepin (8) and 4,5-benzoxepin (11) were synthesized and purified according to the literature [44,45] using standard air-free techniques. Stock solutions of the oxepins 8 and 11 were prepared in acetonitrile and stored at 80 C for use in enzymatic studies. All intermediates − ◦ and final products were characterized by NMR. 1H NMR and 13C NMR spectra were acquired with a Varian UnityINOVA 500 NMR (Varian, Palo Alto, CA, USA) or Varian Mercury 400 BB NMR (Varian, Palo Alto, CA, USA). Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS) unless otherwise noted and coupling constants (J values) are in Hertz (Hz). Infrared spectra were recorded on a Thermo Fischer Scientific Nicolet iS10 FTIR spectrometer (ThermoFischer Scientific, Waltham, MA, USA). UV–vis data were acquired on a Varian Cary 5E UV–vis-NIR spectrophotometer. NADPH and magnesium dichloride were purchased from Sigma Aldrich (St Louis, MO, USA). Potassium phosphate buffer (0.5 M), pooled human liver microsomes, and cytochrome P450 isoforms 1A2, 3A4, and 2E1 were purchased from Corning Life Sciences in Woburn, MA, USA. MilliQ water was used in all incubations, which were carried out in a 37 ◦C water bath. All organic extracts of incubation reactions in dichloromethane were characterized using a Shimazdu GCMS-QP2010 (Shimazdu, Kyoto, Japan) equipped with a 30.0 m SHRX1-5MS column (thickness: 0.25 um, diameter: 0.25 um) and 70 eV electron impact detector. The injector temperature (splitless injection) was held at 250 ◦C and the detector at 260 ◦C. The column temperature increased from 45 ◦C to 250 ◦C at 15 ◦C/min and was held at 250 ◦C for 0 min, after a solvent delay of 5.5 min. The flow rate was 1 mL/min and the total run time was 14.67 min. Standards of the reactants and expected metabolites (see Scheme S1) were prepared in dichloromethane, and retention times and mass spectra were recorded for comparison.

4. Conclusions The incubation (P450 isoforms and pHLM) experiments employing 4,5-benzoxepin (11) clearly indicate epoxidation to form the reactive intermediate 2,3-epoxyoxepin as the major pathway. A lesser metabolic product is the dimeric molecule 20, a reaction product of the oxidation of 11 by CAN. There was no detectable quantity of 21, the other CAN product. These observations suggest that cytochrome P450 epoxidizes the oxepin as its major pathway but also catalyzes consecutive one-electron oxidations. Incubation of 2,7-dimethyloxepin with P450 isoforms and pHLM did not produce the anticipated ring-opened diketone. Instead, dimethylphenols and dimethyldihydroxybenzenes were found. Although these results are essentially consistent with the results of Stok et al. [44], who were the first to identify an oxepin as a minor product of incubation of t-butylbenzene with cytochrome P450, it must be noted that this was bacterial P450 rather than mammalian. In contrast to 4,5-benzoxepin, where the naphthalene oxide isomer is energetically inaccessible, the 1,2-dimethylbenzene oxide isomer is only a few kcal/mol higher in energy than 2,7-dimethyloxepin. While it is not self-evident how to differentiate the formation of muconaldehyde from benzene oxide versus oxepin, the work of Golding and colleagues (e.g., Scheme3) on these tautomers and methyl- and dimethyl-substituted derivatives provides subtle stereochemical probes into these questions. The present work, relying on a “non-natural” metabolite of naphthalene suggests that, for this system, at least, the epoxidation of an oxepin to form a reactive 2,3-epoxyoxepin intermediate is an important, if not dominant, metabolic pathway. Molecules 2020, 25, 4542 9 of 12

Supplementary Materials: The following are available online: characterization data and spectra and analysis of incubation products. Author Contributions: Conceptualization, A.G., H.M.W.-G. and R.W.F.; methodology, A.G, H.M.W.-G., R.W.F. and N.A.C.; investigation, H.M.W.-G., R.W.F. and N.A.C.; writing—original draft preparation, A.G and H.M.W.-G.; writing—review and editing, A.G., H.M.W.-G., R.W.F. and N.A.C.; supervision, A.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: We gratefully acknowledge the help of Professor Stacia Sower and her student Tim Marquis in providing the facilities and instruction for the incubation of substrates with enzymes and to Professor Sterling Tomellini for access to the gas chromatography/mass spectrometry apparatus. We are also grateful for the financial support of the University of New Hampshire Graduate School (Summer TA fellowship—HMG) and the Department of Chemistry at UNH (Daggett Award—RWF). We are thankful for access to instruments (NMR, FTIR, and UV–vis) within the University Instrumentation Center (UIC) at the University of New Hampshire in Durham, NH. We would like to thank Sebastian A. Pantovich for performing the headspace analysis. Finally, we acknowledge David Ashline for his help in obtaining the ESI-MS spectra and for providing his expertise. Conflicts of Interest: The authors declare no conflict of interest.

Appendix A

Synthetic Procedures and Characterization

Preparation of 4,5-Benzoxepin (11) via Autooxidation A 2% solution of 1,4-dihydro-1,4-epoxy-naphthalene (3.0 g) in absolute ethanol (150 mL) was prepared and divided into three quartz tubes [45]. The tubes were sealed and flushed with nitrogen. The reaction mixture was irradiated at 2537 Å for 24 h, then concentrated and purified by column chromatography (100% hexanes). Compound 11 eluted first and was collected in a 6% yield as a yellow 1 solid. H NMR (400 MHz, CDCl3) δ = 5.06 (dd, 2H, J = 7.70, 3.99 Hz), 5.67 (dd, 2H, J = 7.38, 3.98 Hz), 13 6.61–6.66 (AA’XX’, 2H), 6.88-6.92 (AA’XX’, 2H). C NMR (100 MHz, CDCl3) δ = 146.2, 136.0, 129.2, 127.9, 113.4. EI-MS (DCM) = 144 (85), 115 (100), 89 (35). UV–vis (0.2 mg/mL in DCM) λmax = 310.2 nm.

Preparation of 2,7-Dimethyloxepin (8) 2,7-dimethyloxepin was prepared according to the literature procedure published by Paquette and Barrett [46]. Oxidation of 11 with DMDO. The NMR scale reaction was carried out according to the procedure by Nauduri and Greenberg [29].

Preparation of 20 Cerium ammonium nitrate (2 eq, 1.08 g, 1.9 mmol) was dissolved in acetone (15 mL) in a foil-covered 50 mL round bottom flask with stirring. The flask as then cooled to 78 C, and 4,5-benzoxepin (1 eq, − ◦ 136 mg, 0.94 mmol) dissolved in acetone (10 mL) was added dropwise. The reaction was monitored by TLC (3:1 hexanes:ether). After 10 min, the reaction was brought to room temperature and diluted with ether. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (60 mesh silica gel, 6:1 hexanes: EtOAc) and the second fraction was collected and characterized as follows. 1H NMR (400 MHz, CDCl3) δ = 5.79 (d, 1H, J = 8.17 Hz), 6.23 (d, 1H, J = 5.24 Hz), 6.36 (d, 1H, J = 8.17 Hz), 7.01 (d, 1H, J = 5.23 Hz), 7.30 (d, 1H, J = 7.65), 7.31 (t, 1H, J = 7.50 Hz), 7.40 (d, 1H, J = 7.21 Hz), 7.46 (t, 1H, 13 J = 7.55 Hz). C NMR (125 MHz, CDCl3) δ = 79.2, 92.4, 107.5, 126.2, 127.0, 129.1, 129.7, 130.4, 133.8. 1 1 1 1 1 138.8. IR (ATR, neat) = 807 cm− (C-H), 843 cm− (C-H), 1270 cm− (C-O), 1284 cm− (C-O), 1652 cm− (C=C-O). UV–vis (0.2 mg/mL in DCM) λmax = 233, 266, 288 nm. Molecules 2020, 25, 4542 10 of 12

Oxidation of 11 with CAN Preparation of 21 The reaction was carried out according to the procedure by Golding et al. [30]. Cerium ammonium nitrate (2 eq, 1.08 g, 1.9 mmol) was dissolved in acetone (15 mL) in a foil-covered 50 mL round bottom flask with stirring. The flask as then cooled to 78 C, and 4,5-benzoxepin (1 eq, 136 mg, − ◦ 0.94 mmol) dissolved in acetone (10 mL) was added dropwise. The reaction was monitored by TLC (3:1 hexanes:ether). After 1.5 h, the reaction was brought to room temperature and diluted with ether. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (60 mesh silica gel, 9:1 hexanes: 1 EtOAc) and the first fraction was collected and characterized as follows. H NMR (400 MHz, CDCl3) δ = 5.94 (d, 1H, J = 7.53 Hz), 6.48 (d, 1H, 7.52 Hz), 6.66 (s, 1H), 7.35 (d, 1H, J = 7.79 Hz), 7.40 (td, 1H, J = 7.62, 1.18 Hz), 7.62 (td, 1H, J = 7.75, 1.47 Hz), 8.00 (dd, 1H, J = 7.88, 1.46 Hz). 13C NMR (125 MHz, 1 CDCl3) δ = 98.1, 111.4, 127.4, 130.3, 130.8, 133.1, 134.2, 134.4, 140.4, 183.8. IR (ATR, neat) = 797 cm− 1 1 1 ((Ar)-C-H), 1272 cm− (C-O), 1666 cm− (C=O) 2928 cm− (C-H). ESI-MS (dichloromethane) = 147 (100), 131 (50), 119 (47). EI-MS (DCM) = 146 (65), 118 (100), 90 (65). UV–vis (0.2 mg/mL in DCM) λmax = 273.0, 338.8 nm.

General Enzyme Incubation Procedure The quenched reaction mixture was centrifuged (21,000 g) for 4 min to precipitate the pellet. × The supernatant was transferred to a fresh tube and 150 uL of DCM was added. The tube was gently The substrates (300 uM) were incubated in 1.7 mL Eppindorf tubes. A 500 uL reaction mixture containing 100 mM buﬀer, 3.3 mM MgCl2, 1 mM NADPH in 0.5 M NaOH, and 300 uM substrate was preincubated for 5 min in a 37 ◦C water bath. The reactions were initiated by the addition of P450 isoforms (35 pmol/mL) or pHLM (0.5 mg/mL). After inverting the capped tubes twice, the reaction mixtures were incubated for 30 min to 1 h (depending on the enzyme and substrate). The reactions were terminated by the addition of 100 uL acetonitrile. shaken then centrifuged (21,000 g) for 3 min. × The lower organic layer was removed with a micropipette and placed in a fresh tube. The aqueous layer was extracted twice more with DCM (150 uL each). The organic layer (2 uL) was injected into the GC-MS for analysis using the conditions above.

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Sample Availability: Samples of the compounds are not available from the authors.

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