Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine.


Introduction
Although the link between aromaticity and resulting molecular stability is well established, enzymes have a remarkable capacity for dearomatization of many stable and recalcitrant arene and heteroarene substrates. Enzyme-catalyzed dearomatization reactions have been reported under both oxidative and reductive conditions. Examples of redox enzymatic dearomatization reactions of benzene rings A (R = H, Me, COSCoA) that give conjugated cyclohexadiene metabolites include: (i) monooxygenase (MO)-catalyzed epoxidation to yield an arene oxide B (R = H) [1], (ii) ring-hydroxylating dioxygenase (DO)-catalyzed cis dihydroxylation to yield a cis-dihydrodiol C (R = Me) [2], (iii) reductase (CoARed)-catalyzed reduction to give a dihydroarene D (R = COSCoA) (Scheme 1a) [3].
Attempted chemical approaches to the oxidative dearomatization reactions of carbocyclic arenes, shown in Scheme 1a, often result in further oxidation, or rearomatization of the initial products. Due to their instability, relatively few arene oxide metabolites B have been isolated from monooxygenase (MO)-catalyzed epoxidations of substituted benzene substrates A. Ring-hydroxylating dioxygenase (DO)-catalyzed cis-dihydroxylation of similar substrates A can, however, produce more stable cis-dihydrodiols C. Despite their The ability of similar oxidative dearomatization reactions of a thiophene substrate E to replicate MO-catalyzed epoxidation to yield epoxide G, DO-catalyzed cis-dihydroxylation to give cis-dihydrodiol H, and heteroatom oxidation to give sulfoxide F (Scheme 1b) using either type of oxygenase enzyme is examined in this review.
Aromaticity of the monocyclic six-membered arenes, e.g., benzene, and five-membered heteroarenes, e.g., pyrrole, thiophene, and furan, confers extra stability and depends on the descriptor used [10]. Resonance energies have been widely used as an indicator of decreasing aromaticity in a sequence, from the most stable benzene > thiophene > pyrrole > furan. Most other qualitative and quantitative descriptors of aromaticity also suggest that the sequence should be: benzene > thiophene > pyrrole > furan [11].
As acceptable thiophene substrates for ring-hydroxylating dioxygenases are generally smaller than for monooxygenase substrates, the topic of dioxygenase-catalyzed cis-dihydroxylation and sulfoxidation of thiophene substrates is initially considered. The only cis-dihydrodiol metabolites from the monocyclic five-membered heteroarenes, thiophene, pyrrole, and furan, of sufficient stability to be isolated and characterized are derived from thiophene substrates. Thus, the relatively stable cis-dihydrodiol metabolite H was obtained from toluene dioxygenase (TDO)-catalyzed oxidation of thiophene E along with the unstable thiophene-S-oxide F (Scheme 1b) [12].
Early studies of the bacterial metabolism of thiaarenes also showed that dioxygenasecatalyzed cis-dihydroxylation of carbocyclic and heterocyclic rings could yield chiral cisdihydrodiols as transient metabolites. Furthermore, isolation of these intermediates was possible when using P. putida mutant strains (or E. coli recombinant strains), expressing dioxygenases where an enzyme that is usually present in the metabolic pathway, a cisdihydrodiol dehydrogenase was blocked (or absent) [26][27][28].
The results in Table 1 show that TDO-catalyzed sulfoxidation of thiophenes 1a-g is the major dearomatization pathway, with cis-dihydroxylation of thienyl and phenyl rings, respectively, being minor pathways. CYP450-catalyzed oxidation of thiophenes 1a, 1b, and 1g yielded the corresponding sulfoxides 6a, 6b, and 6g, and their dimers 7a, 7b, and 7g, as major products, but deoxygenation of dimers was not reported (Scheme 2).

Molecular Docking of Thiophenes 1a and 1g at the TDO Active Site
The Autodock Vina program was used for docking toluene A (R = Me, Scheme 1a) and other arene and heteroarene substrates within the active site of toluene dioxygenase [71,72]. An X-ray crystal structure of the TDO showed the toluene substrate bound at the active site by proximate amino acids but without dioxygen complexed to Fe(III) [73]. An X-ray crystal structure of NDO displayed dioxygen coordinated with Fe(III) and indole substrate bonded at the active site [74]. The O 2 -Fe(III) complex of NDO was then inserted into the TDO active site employing the reported procedure [72]. The interaction between dioxygen and Fe(III) during the catalysis of the cis-dihydroxylation is considered to involve a hydroperoxide (Fe-OOH) [29]. Attractive interactions between substrates and proximate amino acids (Phe-216, His-222, Ile-276, Leu-272, Ile-324, Val-309, Leu-272, Figure 1A) allow the preferred binding orientation to be predicted; it matched both with the regio-and stereo-selectivity observed during TDO-catalyzed cis-dihydroxylation of toluene and other substituted benzene substrates to form cis-dihydrodiols (Scheme 1a) [72,[74][75][76][77][78][79].
The results in Table 1 show that TDO-catalyzed sulfoxidation of thiophenes 1a-g is the major dearomatization pathway, with cis-dihydroxylation of the thienyl and phenyl rings, respectively, being the minor pathways. CYP450-catalyzed oxidation of thiophenes 1a, 1b, and 1g yielded the corresponding sulfoxides 6a, 6b, and 6g, and their dimers 7a, 7b, and 7g, as major products, but deoxygenation of dimers was not reported (Scheme 2).

Molecular Docking of Thiophenes 1a and 1g at the TDO Active Site
The Autodock Vina program was used for docking toluene A (R = Me, Scheme 1a) and other arene and heteroarene substrates within the active site of toluene dioxygenase [71,72]. An X-ray crystal structure of the TDO showed the toluene substrate bound at the active site by proximate amino acids but without dioxygen complexed to Fe(III) [73]. An X-ray crystal structure of NDO displayed dioxygen coordinated with Fe(III) and indole substrate bonded at the active site [74]. The O2 Fe(III) complex of NDO was then inserted into the TDO active site employing the reported procedure [72]. The interaction between dioxygen and Fe(III) during the catalysis of the cis-dihydroxylation is considered to involve a hydroperoxide (Fe-OOH) [29]. Attractive interactions between substrates and proximate amino acids (Phe-216, His-222, Ile-276, Leu-272, Ile-324, Val-309, Leu-272, Figure 1A) allow the preferred binding orientation to be predicted; it matched both with the regio-and stereo-selectivity observed during TDO-catalyzed cis-dihydroxylation of toluene and other substituted benzene substrates to form cis-dihydrodiols (Scheme 1a) [72,[74][75][76][77][78][79]. A similar in situ docking approach, applied earlier to other arene and heteroarene substrates [71,72], was used to determine the preferred binding orientations of thiophene 1a ( Figure 1A,B) and 2-phenylthiophene 1g (Figure 2A,B). As expected for a smaller five-membered heteroarene substrate, binding energies (± 0.5 kcal mol −1 ) for thiophene 1a, Figure 1A (−3.63 kcal mol −1 ) and B (−3.3 kcal mol −1 ), were lower compared to a six-membered arene substrate, e.g., toluene A (R = Me, −5.0 kcal mol −1 ). The minimum distances between the nearest oxygen and sulfur atoms (proximity value, 3.8 Å, Figure  1A), or between proximate oxygen atoms and the 2,3-bond (3.0-3.7 Å, Figure 1B), are similar to those found between TDO and toluene A (R = Me). The favored orientation of thiophene 1a in Figure 1A is consistent with TDO-catalyzed sulfoxidation to yield sulfoxide 5a and derived bioproducts 6a and 7a [12]. The reduced substrate size, lower binding energy, but favorable proximity factor for compound 1a in Figure 1B could be factors in the reduced ee value (44%) and lower yield of the isolated heterocyclic cis/trans diols 2a/4a (Table 1). A similar in situ docking approach, applied earlier to other arene and heteroarene substrates [71,72], was used to determine the preferred binding orientations of thiophene 1a ( Figure 1A,B) and 2-phenylthiophene 1g (Figure 2A,B). As expected for a smaller fivemembered heteroarene substrate, binding energies (± 0.5 kcal mol −1 ) for thiophene 1a, Figure 1A (−3.63 kcal mol −1 ) and B (−3.3 kcal mol −1 ), were lower compared to a sixmembered arene substrate, e.g., toluene A (R = Me, −5.0 kcal mol −1 ). The minimum distances between the nearest oxygen and sulfur atoms (proximity value, 3.8 Å, Figure 1A), or between proximate oxygen atoms and the 2,3-bond (3.0-3.7 Å, Figure 1B), are similar to those found between TDO and toluene A (R = Me). The favored orientation of thiophene 1a in Figure 1A is consistent with TDO-catalyzed sulfoxidation to yield sulfoxide 5a and derived bioproducts 6a and 7a [12]. The reduced substrate size, lower binding energy, but favorable proximity factor for compound 1a in Figure 1B could be factors in the reduced ee value (44%) and lower yield of the isolated heterocyclic cis/trans diols 2a/4a ( Table 1). The preferred orientation of 2-phenylthiophene 1g at the TDO active site ( Figure 2A) has a relatively high binding energy (−5.5 kcal mol −1 ) and proximity of the phenyl group to dioxygen (3.4-3.5 Å). The predicted regioselectivity and absolute configuration of cis-dihydrodiol 8g was found, experimentally, to be (1S,2R) ( Table 1) [12].
The preferred orientation of substrate 1g, at the TDO active site, predicted from the binding energy (−5.3 kcal mol −1 ) and the distance of the sulfur to dioxygen (2.9 Å), should yield (1S)-sulfoxide 5g. However, the low inversion barrier, predicted for sulfoxide 5g ΔG ≠ 11.2 kcal mol −1 ) [64], indicated that it would rapidly racemize, and this was confirmed by the isolation of racemic dimer 6g ( Table 1). The docking of substrate 1g did not present an orientation that would lead to cis-dihydrodiol metabolite 2g, and none of the possible cis/trans-dihydrodiols 2b-g/4b-g were isolated (Table 1) [12].
The preferred orientation of substrate 1g, at the TDO active site, predicted from the binding energy (−5.3 kcal mol −1 ) and the distance of the sulfur to dioxygen (2.9 Å), should yield (1S)-sulfoxide 5g. However, the low inversion barrier, predicted for sulfoxide 5g ∆G = 11.2 kcal mol −1 ) [64], indicated that it would rapidly racemize, and this was confirmed by the isolation of racemic dimer 6g ( Table 1). The docking of substrate 1g did not present an orientation that would lead to cis-dihydrodiol metabolite 2g, and none of the possible cis/trans-dihydrodiols 2b-g/4b-g were isolated (Table 1) [12].
Ab initio computational and quantum chemistry studies predicted that enantiomers of benzo[b]thiophene sulfoxide 23a would undergo pyramidal inversion and racemization at ambient temperature with predicted inversion barriers of (∆G = = 22.9-23.9 kcal mol −1 ) [64,65]. Experimental validation was finally provided by the isolation and observed racemization of the (1R)-benzo[b]thiophene sulfoxide 23a obtained by a styrene monoxygenase-catalyzed sulfoxidation of substrate 10a (Section 6.3). Further confirmation of predicted inversion barriers was provided by racemization of (1R)-enantiomers of sulfoxides 23b and 23c, at ambient temperature. This occurred at a slower rate over a 24 h period, and kinetic studies provided pyramidal inversion barriers(∆G = ) of 25.1 and 26.4 kcal mol −1 , respectively, [83]. Similar inversion barriers are expected for sulfoxide metabolites formed from CYP450-catalyzed sulfoxidation of benzo[b]thiophene-containing drugs, e.g., zileuton 10e and brexpiprazole 10g (Section 6).
The angular junction between four fused benzene rings in benzo[c]phenanthrene is classified as a fjord region, and BPDO-catalyzed cis-dihydroxylation occurred, exclusively, at this region [94]. The region between fused benzene, thiophene, and naphthalene rings, found in benzo[b]naphtho[1,2-d]thiophene 30a, is assigned as a pseudo-fjord region.
Ring-hydroxylating dioxygenases with smaller active sites, e.g., TDO, normally only catalyze cis-dihydroxylation of mono-and bi-cyclic arenes [4][5][6][7][8][9]. The inability of its active site to accommodate many larger polycyclic thiophenes, e.g., tetracyclic substrates 30a and 34, allied to their limited aqueous solubility, are major factors in their resistance to metabolism. The larger active sites of NDO and BPDO are of similar structure (44% sequence identity) [95,96], and the wider entrance to the BPDO active site makes it is among the most successful dioxygenases for binding and biodegrading tri-, tetra-, and penta-cyclic arenes and thiaarenes.
Autodock Vina molecular docking studies of dibenzo[b,d]thiophene 33 as substrate led to the unexpected prediction that it could accommodate a tricyclic substrate within the relatively small active site of TDO [79]. Furthermore, it was predicted that the preferred in vitro orientation of dibenzo[b,d]thiophene 33 would result in the formation of (1R,2S)-dihydrodiol 35. This proposition was validated in vivo, when TDO-catalyzed (P. putida UV4) cis-dihydroxylation occurred within the pseudo-bay region, to give exclusively (1R,2S)-dihydrodiol 35 (>98% ee, 16% isolated yield) [79].
A more polar minor metabolite, resulting from a further cis-dihydroxylation within the alternative pseudo-bay region, was identified as bis-cis-(1R,2S,7R,8S)-tetrahydro-diol 49. Confirmation for this metabolic sequence (Scheme 8) was obtained by using cis-dihydrodiol metabolite 48 as a substrate, and, further, cis-dihydroxylation yielded cis-tetraol 49. Metabolite 49 was the first identified member of the heteroarene bis-cis-dihydrodiol series but a similar type of BPDO-catalyzed tetrahydroxylation was observed when using chrysene as a substrate, which also contained two bay regions [93].

Application of Thiophene cis-Dihydrodiols in Thiophene Epoxide Synthesis
Enantiopure cis-dihydrodiols, derived from enzymatic cis-dihydroxylation of substituted arene substrates, have been widely used as precursors in the chemoenzymatic synthesis of many chiral natural products and other types of metabolites, including arene oxides and trans-dihydrodiols [4][5][6][7][8][9]41,71]. While few cis-dihydrodiol metabolites of heteroarene substrates have been utilized in synthesis, a small number of relatively stable bicyclic metabolites, derived from quinoline, 2-chloroquinoline, and 4-chloroquinoline, have proved to be useful chiral synthons. Thus, chemoenzymatic syntheses of chiral A more polar minor metabolite, resulting from a further cis-dihydroxylation within the alternative pseudo-bay region, was identified as bis-cis-(1R,2S,7R,8S)-tetrahydrodiol 49. Confirmation for this metabolic sequence (Scheme 8) was obtained by using cis-dihydrodiol metabolite 48 as a substrate, and, further, cis-dihydroxylation yielded cis-tetraol 49. Metabolite 49 was the first identified member of the heteroarene bis-cis-dihydrodiol series but a similar type of BPDO-catalyzed tetrahydroxylation was observed when using chrysene as a substrate, which contained two bay regions [93].
The paucity of reports on the synthetic applications of thiophene sulfoxides and cisdihydrodiols is possibly due to their unavailability and perceived instability. Monocyclic thiophene sulfoxides, although generally considered as unstable, can be stabilized by the presence of bulky substituents. More stable sulfoxides of this type have been used to determine sulfoxide inversion barriers by spectroscopic methods [68,69].
A further example of the synthetic application of thiophene sulfoxides is provided by the reaction of the relatively stable 3,4-di-tert-butylthiophene-1-oxide with dimethylacetylene dicarboxylate. This formed an unstable cycloadduct that decomposed spontaneously, providing a source of singlet sulfur monoxide [113]; its reaction with dienes and alkynes delivered a useful synthetic route to thiirane and thiirene oxides.
To test the potential application of thiophene cis-dihydrodiols in chemoenzymatic synthesis, it was important to select a relatively stable metabolite. The stability of cisand trans-dihydrodiol metabolites 17a and 19a, formed by dioxygenase-catalyzed cisdihydroxylation of benzo[b]thiophene 10a (Scheme 4), prompted a study of their use in the synthesis of a thiophene epoxide.
The three-step reaction sequence in Scheme 9 started from an isomeric mixture of metabolites 17a and 19a, proceeded via dioxoles 50, and trans-chloroacetate 51 as intermediates and yielded benzo[b]thiophene-2,3-oxide 52. A similar sequence was used in the synthesis of K-region arene oxides [114]. Epoxide 52 was identified by NMR spectroscopy (THF-d 8 ) and MS analysis. On attempted chromatographic purification, it isomerized to a mixture of 3-hydroxybenzo[b]thiophene 53 and the keto tautomer 54 [82]. Int spontaneously, providing a source of singlet sulfur monoxide [113]; its reaction with dienes and alkynes delivered a useful synthetic route to thiirane and thiirene oxides.
To test the potential application of thiophene cis-dihydrodiols in chemoenzymatic synthesis, it was important to select a relatively stable metabolite. The stability of cis-and trans-dihydrodiol metabolites 17a and 19a, formed by dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophene 10a (Scheme 4), prompted a study of their use in the synthesis of a thiophene epoxide.

CYP450-Catalyzed Epoxidation of Monocyclic Thiophenes
Both benzene oxide and benzene cis-dihydrodiol metabolites have been chemi synthesized and characterized. Scheme 1a illustrates that CYP450-catalyzed epoxida of mono-and poly-cyclic arene substrates [124,125] can provide an alternative oxida dearomatization pathway compared to dioxygenase-catalyzed cis-hydroxylation. T major enzymatic dearomatization pathways (sulfoxidation, cis-dihydroxylation, epoxidation) are possible for mono-and poly-cyclic thiophene substrates (Scheme 1b An important difference between cis-dihydroxylation and epoxidation of thioph is that thiophene cis-dihydrodiols have been isolated and fully characterized whil date, thiophene epoxide metabolites have not. It is important to consider what evid is available for the involvement of transient benzene oxide metabolites prior to thei Scheme 10. CYP450-catalyzed sulfoxidation of thiophenes 1a, g, k, m, p, q, s, t, v, z. Cytochrome P450-catalyzed oxidation of the monosubstituted thiophene-containing drugs, tienilic acid isomer 1m, methapyrilene 1s, and the disubstituted thiophenes ticlopidine 1p, clopidogrel 1q, and OSI-930 1t was found to give transient sulfoxide metabolites 5m,s,p,q, and 5t, respectively, that rapidly dimerized to 6m,s,p,q,t (Scheme 10 and Figure 5) [57,121,122]. In the presence of NMM in the incubation, cyclo-adducts 55m,p,q, and 55s were obtained at the cost of the thiophene sulfoxide dimers [120]. Incubation of tri-substituted thiophene dimethenamid-P ( Figure 5) herbicide with basidiomycete Irpex consors produced a stable thiophene sulfoxide and two isomeric 2-thiolenones [123].
An important difference between cis-dihydroxylation and epoxidation of thiophenes is that thiophene cis-dihydrodiols have been isolated and fully characterized while, to date, thiophene epoxide metabolites have not. It is important to consider what evidence is available for the involvement of transient benzene oxide metabolites prior to their detection, isolation, and synthesis when attempting to identify transient thiophene epoxide metabolites.
Other thiophene-containing drugs, tiquizium bromide 1w, morantel 1x, and tenoxicam 1y, were also oxidized in microsomal incubations in the thiophene ring, but the position of oxidation was not determined [132][133][134]. For two thiophene-containing drugs, duloxetine and eprosartan, and one benzothiophene-containing drug, raloxifene, metabolic oxidation of the thiophene ring was researched and not detected, other parts of the molecules being oxidized [57].
Following the report of an unusual rearrangement reaction of arenes during enzymatic aromatic hydroxylation, the NIH Shift, this observation was widely used as strong evidence for the intermediacy of transient arene oxides [125,135]. The NIH Shift requires migration of an atom (e.g., D or T) or group and retention at an adjacent site during aromatic hydroxylation of substituted arenes. A similar migration and retention of label Scheme 11. CYP450-catalyzed oxidation of thiophenes 1a,g,k,l-u,z.
Other thiophene-containing drugs, tiquizium bromide 1w, morantel 1x, and tenoxicam 1y, were also oxidized in microsomal incubations in the thiophene ring, but the position of oxidation was not determined [132][133][134]. For two thiophene-containing drugs, duloxetine and eprosartan, and one benzothiophene-containing drug, raloxifene, metabolic oxidation of the thiophene ring was researched and not detected, other parts of the molecules being oxidized [57].
Following the report of an unusual rearrangement reaction of arenes during enzymatic aromatic hydroxylation, the NIH Shift, this observation was widely used as strong evidence for the intermediacy of transient arene oxides [125,135]. The NIH Shift requires migration of an atom (e.g., D or T) or group and retention at an adjacent site during aromatic hydroxylation of substituted arenes. A similar migration and retention of label was observed during TDO-catalyzed arene cis-dihydroxylation and dehydration of the D-labelled cisdihydrodiol metabolites to give phenols [136]. In addition to these two mechanisms for the NIH Shift, other mechanisms have since been reported [137]. The NIH Shift was, however, not observed during CYP450-catalyzed oxidation of thiophenes 1a,g,k,l during formation of the corresponding hydroxythiophene metabolites 59a,g,k,l and, therefore, could not be used as evidence for epoxidation.
Arenes can be oxidized by dioxygen and CYP450s forming arene oxides that are then enzymatically hydrolyzed by microsomal epoxide hydrolase (mEH) to give stable trans-dihydrodiols. Arene oxides are relatively stable and have been produced by either chemical or enzymatic synthesis. Thiophene trans-dihydrodiols 4a,h-k have been isolated from TDO-catalyzed oxidations of thiophenes as epimers of the initially formed cis-dihydrodiols 2a,h-k [12]; they were relatively stable and did not readily dehydrate. Thus, it was expected that trans-dihydrodiols could be formed from thiophene epoxides 58a,g,k,l, during liver microsomal incubations, as presented in Scheme 11. Neither transdihydrodiols 4a,g,k,l, nor the corresponding cis epimers 2a,g,k,l, have been detected as metabolites during CYP450-catalyzed oxidation of any monocyclic thiophenes including compounds 1a,g,k,l [54,57,128].
Why were thiophene trans-dihydrodiols not found? One possibility would be that they are formed below the level of detection. Quantum chemical analysis results predicted that the energy barrier to direct hydrolysis of a thiophene epoxide by water was too high [122]. However, since arene oxide hydrolysis requires catalysis by mEH, a similar outcome might be expected for thiophene epoxides. The size of the active site of mEH is able to accommodate arene oxide sizes from one aromatic ring up to five fused rings and aliphatic epoxides and, thus, should accept small thiophene epoxides. The hydrophobicity of thiophene epoxides should be close to that of arene oxides of similar size. Maybe mEH is not the appropriate enzyme or the half-life of thiophene epoxides is too short for being transferred to the catalytic site of mEH. Despite some thiophenes and thiophene-containing drugs having similar types of substituents, no trans-dihydrodiols nor their cis epimers have been yet detected directly during microsomal incubations.
The stability of thiophene epoxides [122] could be increased using the same approach adopted for arene oxides [135] that were stabilized by the presence of bulky or electronwithdrawing substituents [137,138]. Despite some model thiophenes and thiophenecontaining drugs having similar types of substituents, no epoxide metabolites have been detected directly.
Relatively few examples of hydrate formation during mono-or di-oxygenase-catalyzed oxidations of arenes or heteroarenes are available . Metabolites 64g, 64r, and 64z are rare examples of heteroarene hydrates formed through CYP450-catalyzed epoxidation of the corresponding thiophenes 1g,r, and 1z. [140]. Monohydroxylation (CYP450) at allylic or benzylic positions of dihydroarenes yielded polycyclic arene hydrates [141]. Dioxygenasecatalyzed cis-dihydroxylation of monosubstituted phenols also produced hydrates, and their keto tautomers, as minor metabolites [78]. The formation of thiophene hydrates and phenol hydrates, during oxidative biotransformations of the corresponding phenol and thiophene substrates, could be regarded as dearomatizations. However, in each case, several steps are involved rather than direct hydration reactions.
Monocyclic thiophene epoxides and sulfoxides have also been identified as biologically reactive intermediates, that react in situ with the enzyme responsible for their production. Several thiophene-containing drugs have been identified as mechanism-based inactivators of cytochrome P450s and to react with the apoenzyme [142][143][144][145][146].
Monocyclic thiophene epoxides and sulfoxides have also been identified as biologically reactive intermediates, that react in situ with the enzyme responsible for their production. Several thiophene-containing drugs have been identified as mechanism-based inactivators of cytochrome P450s and to react with the apoenzyme [142][143][144][145][146].
No convincing evidence was found for epoxide hydrolase (EH)-catalyzed hydrolysis of thiophene epoxide metabolites, yielding trans-dihydrodiols from metabolism of thiophenes and thiophene-containing drugs (Scheme 11).
Later studies of CYP450-catalyzed metabolism of tetracyclic thiophene 34 also identified sulfoxide 74 and sulfone 75; in addition, two trans-dihydrodiols, derived from the transient arene oxides 70 and 71 and two phenols, resulting from undetected arene oxides 72 and 73, were identified (Scheme 14) [109]. The proposed CYP450-catalyzed oxidations shown in Scheme 14 involve five enzymatic dearomatization reactions during production of one sulfoxide 74 and four arene oxide intermediates 70-73.

Monooxygenase-Catalyzed Thiophene Ring Oxidation of Thienopyridine Prodrugs
Some important antiplatelet and antiaggregant prodrugs, with a tetrahydro-thienopyridine structure 76 (Ticlopidine 1p, Clopidogrel 1q, Figure 5 and Scheme 15), are metabolized in mammals by a series of reactions on the thiophene ring, leading to a thiol acid derivative that inhibits the G-protein ADP receptor P2Y12 [157]. This family of compounds was discovered in 1978 [158], the receptor was located in 2000 and the complex formation of the active metabolite was deciphered during 2009-2013 [158]. Int Later studies of CYP450-catalyzed metabolism of tetracyclic thiophene 34 also identified sulfoxide 74 and sulfone 75; in addition, two trans-dihydrodiols, derived from the transient arene oxides 70 and 71 and two phenols, resulting from undetected arene oxides 72 and 73, were identified (Scheme 14) [109]. The proposed CYP450-catalyzed oxidations shown in Scheme 14 involve five enzymatic dearomatization reactions during production of one sulfoxide 74 and four arene oxide intermediates 70-73.

Monooxygenase-Catalyzed Thiophene Ring Oxidation of Thienopyridine Prodrugs
Some important antiplatelet and antiaggregant prodrugs, with a tetrahydro-thienopyridine structure 76 (Ticlopidine 1p, Clopidogrel 1q, Figure 5 and Scheme 15), are metabolized in mammals by a series of reactions on the thiophene ring, leading to a thiol acid derivative that inhibits the G-protein ADP receptor P2Y12 [157]. This family of compounds was discovered in 1978 [158], the receptor was located in 2000 and the complex formation of the active metabolite was deciphered during 2009-2013 [158].
The important role played by CYP450 and unspecific fungal peroxygenase (UFO)-catalyzed epoxidation and sulfoxidation is exemplified by the thienopyridine drug metabolism route in Scheme 15. Further reactions of the unstable epoxide and sulfoxide metabolites led to the formation of many other products, including sulfoxide dimers, hydroxythiophenes, thiolactones, sulfenic acids, and thiols. One of the first steps in the metabolism of thienopyridines 76, clopidogrel 1q, and ticlopidine 1p was oxidation of the thiophene ring by a CYP450 monooxygenase (or UFO peroxygenase) to yield an unstable thiophene epoxide 77. This intermediate isomerized into a 2-hydroxythiophene 78 and its thiolactone tautomers 79a and 79b (Path A, Scheme 15) [159]. Further CYP450-or peroxygenase-catalyzed oxidation of tautomers 78/79a/79b Scheme 15. CYP450-or UFO-catalyzed oxidative metabolism of thienopyridine antiplatelet compounds 76 (ticlopidine 1p and clopidogrel 1q).
The important role played by CYP450 and unspecific fungal peroxygenase (UFO)catalyzed epoxidation and sulfoxidation is exemplified by the thienopyridine drug metabolism route in Scheme 15. Further reactions of the unstable epoxide and sulfoxide metabolites led to the formation of many other products, including sulfoxide dimers, hydroxythiophenes, thiolactones, sulfenic acids, and thiols.
Some metabolites of the 2-substituted thiophene razuprotafib 1v, including a methylthioether, may also be formed by Path A with Path B being also involved in formation of other metabolites [170,171]. Metabolic reactions of thiophenes catalyzed by cytochrome P450 were reviewed recently [172].

Conclusions
A major emphasis of this review has been on the complementary nature of monooxygenase and dioxygenase enzyme activities, in the context of oxidative dearomatization of mono-and poly-cyclic thiophenes. Monooxygenase enzymes, expressed by mammalian, fungal, and bacterial cells, were used in the oxidative aromatization of a wider range of types and sizes of thiophenes compared with ring-hydroxylating dioxygenases.
Wild-type bacteria, expressing ring hydroxylating dioxygenase enzymes, have been employed to metabolize and remove thiophenes from fossil fuels. Mutant and recombinant bacterial strains, expressing these enzymes, were utilized to intercept and scale up the production of thiophene sulfoxide and dihydrodiol metabolites from relatively small thiophene substrates. Factors influencing chemo-, regio-, and stereo-selectivity, stability and mechanisms of dioxygenase-catalyzed oxidations of thiophenes are discussed.
Enantioenriched thiophene sulfoxide metabolites were used to determine inversion barriers and compare with predictions from calculated values. These barriers are of potential importance within the context of individual sulfoxide enantiomers from drugs having different efficacies. A range of inversion barriers were predicted for sulfoxide metabolites obtained during CYP450-catalyzed oxidation of thiophene-containing drugs. The application of a thiophene cis-dihydrodiol metabolite in the first synthesis a thiophene epoxide suggests that more stable thiophene epoxides could be obtained using this method.
Lessons can be learned from comparisons of mono-and di-oxygenase-catalyzed oxidation of arenes with thiophenes where metabolite instability is often a major problem. Factors to address this include the use of bulky or electron-withdrawing substituents or benzo-fusion to stabilize thiophene sulfoxides and epoxides. Factors found to influence the thermal stability and dynamic stereochemistry (racemization, cis/trans isomerization) of metabolites, isolated from dioxygenase-catalyzed oxidation of thiophenes, are also applica-ble to those derived from monooxygenases. Metabolism of carbocyclic arenes by dioxygenases can yield cis-dihydrodiols while transient arene oxides produced by monooxygenases hydrolyze to isolable trans-dihydrodiols. Possible reasons are discussed for the almost reverse scenario, where dioxygenases catalyze formation of thiophene trans-dihydrodiols as major metablites under aqueous condations while monooxygenases do not.
Further research using suitably substituted thiophene substrates with appropriate dioxygenases could produce cis-dihydrodiol metabolites for use in the synthesis of more stable thiophene epoxides and sulfoxides. Monooxygenase-catalyzed production of transient thiophene epoxide metabolites in the presence of a range of epoxide hydrolases might finally lead to trans-dihydrodiol production. The question of why model benzo[b]thiophenes and benzo[b]thiophene-containing drugs and monooxygenases do not appear to produce either epoxides or trans-dihydrodiols remains unanswered.