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.

Toluene Dioxygenase-Catalyzed cis-Dihydroxylation of Quinolines: A Molecular Docking Study and Chemoenzymatic Synthesis of Quinoline Arene Oxides.

Molecular docking studies of quinoline and 2-chloroquinoline substrates at the active site of toluene dioxygenase (TDO), were conducted using Autodock Vina, to identify novel edge-to-face interactions and to rationalize the observed stereoselective cis-dihydroxylation of carbocyclic rings and formation of isolable cis-dihydrodiol metabolites. These in silico docking results of quinoline and pyridine substrates, with TDO, also provided support for the postulated cis-dihydroxylation of electron-deficient pyridyl rings, to give transient cis-dihydrodiol intermediates and the derived hydroxyquinolines. 2-Chloroquinoline cis-dihydrodiol metabolites were used as precursors in the chemoenzymatic synthesis of enantiopure arene oxide and arene dioxide derivatives of quinoline, in the context of its possible mammalian metabolism and carcinogenicity.

Chemoenzymatic Synthesis of (-)-Ribisins A and B from Dibenzo[b,d]furan.

cis-Dihydrodiols, derived from monocyclic aromatic compounds, are valuable chiral pool intermediates for the synthesis of cyclic natural products. A drawback of this approach, to the synthesis of polycyclic secondary metabolites, is that additional rings must be annulated. To date, relatively few chiral natural products have been synthesized from polycyclic arene cis-dihydrodiols. Fungal metabolites, (-)-ribisins A and B, have now been obtained by functional group manipulation of a tricyclic arene metabolite, obtained from toluene dioxygenase-catalyzed regioselective and stereoselective cis-dihydroxylations of dibenzo[b,d]furan. The synthetic sequences were marginally shorter than the alternative routes, using monocyclic arene cis-dihydrodiols, and required no carbon-carbon bond-forming reactions.

Draft Genome Sequences of Pseudomonas putida UV4 and UV4/95, Toluene Dioxygenase-Expressing Producers of cis-1,2-Dihydrodiols.

Here, we present draft genome sequences of Pseudomonas putida strains UV4 and UV4/95, which demonstrate an ability to conduct a wide range of industrially important biotransformations of arenes, alkenes, and phenols.

Enantiopurity and absolute configuration determination of arene cis-dihydrodiol metabolites and derivatives using chiral boronic acids.

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis-1,2-diol metabolites, including boronate formation, using racemic or enantiopure (+) and (-)-2-(1-methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono- and poly-cyclic arene cis-dihydrodiol, cyclohex-2-en-1-one cis-diol, heteroarene cis/trans-2,3-diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2-[1-methoxy-2,2-dimethylpropyl]phenyl boronic acid (MDPBA) and 2-[1-methoxy-1-phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis-diol metabolites, are presented.

Chemoenzymatic synthesis of enantiopure hydroxy sulfoxides derived from substituted arenes.

Enantiopure ß-hydroxy sulfoxides and catechol sulfoxides were obtained, by chemoenzymatic synthesis, involving dioxygenase-catalysed benzylic hydroxylation or arene cis-dihydroxylation and cis-diol dehydrogenase-catalysed dehydrogenation. Absolute configurations of chiral hydroxy sulfoxides were determined by X-ray crystallography, ECD spectroscopy and stereochemical correlation. The application of a new range of ß-hydroxy sulfoxides as chiral ligands was examined.

Toluene dioxygenase-catalyzed synthesis and reactions of cis-diol metabolites derived from 2- and 3-methoxyphenols.

Using toluene dioxygenase as biocatalyst, enantiopure cis-dihydrodiol and cis-tetrahydrodiol metabolites, isolated as their ketone tautomers, were obtained from meta and ortho methoxyphenols. Although these isomeric phenol substrates are structurally similar, the major bioproducts from each of these biotransformations were found at different oxidation levels. The relatively stable cyclohexenone cis-diol metabolite from meta methoxyphenol was isolated, while the corresponding metabolite from ortho methoxyphenol was rapidly bioreduced to a cyclohexanone cis-diol. The chemistry of the 3-methoxycyclohexenone cis-diol product was investigated and elimination, aromatization, hydrogenation, regioselective O-exchange, Stork-Danheiser transposition and O-methylation reactions were observed. An offshoot of this technology provided a two-step chemoenzymatic synthesis, from meta methoxyphenol, of a recently reported chiral fungal metabolite; this synthesis also established the previously unassigned absolute configuration.

Abiotic methanogenesis from organosulphur compounds under ambient conditions.

Methane in the environment is produced by both biotic and abiotic processes. Biomethanation involves the formation of methane by microbes that live in oxygen-free environments. Abiotic methane formation proceeds under conditions at elevated temperature and/or pressure. Here we present a chemical reaction that readily forms methane from organosulphur compounds under highly oxidative conditions at ambient atmospheric pressure and temperature. When using iron(II/III), hydrogen peroxide and ascorbic acid as reagents, S-methyl groups of organosulphur compounds are efficiently converted into methane. In a first step, methyl sulphides are oxidized to the corresponding sulphoxides. In the next step, demethylation of the sulphoxide via homolytic bond cleavage leads to methyl radical formation and finally to methane in high yields. Because sulphoxidation of methyl sulphides is ubiquitous in the environment, this novel chemical route might mimic methane formation in living aerobic organisms.

Reactions of enantiopure cyclic diols with sulfuryl chloride.

Monocyclic allylic cis-1,2-diols reacted with sulfuryl chloride at 0 °C in a regio- and stereo-selective manner to give 2-chloro-1-sulfochloridates, which were hydrolysed to yield the corresponding trans-1,2-chlorohydrins. At -78 °C, with very slow addition of sulfuryl chloride, cyclic sulfates were formed in good yields, proved to be very reactive with nucleophiles and rapidly decomposed on attempted storage. Reaction of a cyclic sulfate with sodium azide yielded a trans-azidohydrin without evidence of allylic rearrangement occurring. An enantiopure bicyclic cis-1,2-diol reacted with sulfuryl chloride to give, exclusively, a trans-1,2-dichloride enantiomer with retention of configuration at the benzylic centre and inversion at the non-benzylic centre; a mechanism is presented to rationalise the observation.

An evaluation of substituent effects on aromatic edge-to-face interactions and CF-π versus CH-π interactions using an imino torsion balance model.

A selection of imines derived from phenyl t-butyl ketones and substituted 2-phenylethylamines or phenylalanine exhibit slow rotation around the aryl­imino bond at ambient temperature, resulting in a large non-equivalence of the ortho hydrogens in the 1H NMR spectra. This facilitates assessment of aryl substituent effects on the face tilted-T CH­π interaction between a phenyl ring (A) on the imino carbon proximate to the terminal phenyl ring (B). Analysis of the marked temperature dependence of the chemical shift of the interacting ortho hydrogen affords estimates of the opposing enthalpic and entropic factors involved in the rapid equilibrium between the closed edge-to-face conformation and alternative open conformations devoid of a CH­π interaction while in solution. Above ca. 80 °C the entropy term (TΔS) cancels out the enthalpy (ΔH) favouring the closed conformation and open conformations are preferred. Accordingly, commonly reported binding free energies may not be a good measure of the energetic strength of intramolecular aromatic interactions. Investigation of an ortho fluoro substituted compound indicates that a CF­π interaction is at least 1.0 kcal mol−1 weaker in enthalpy than the CH­π interaction. Several X-ray crystal structures depicting an intramolecular edge-to-face interaction are presented.

Chemoenzymatic synthesis of monocyclic arene oxides and arene hydrates from substituted benzene substrates.

Enantiopure cis-dihydrodiol bacterial metabolites of substituted benzene substrates were used as precursors, in a chemoenzymatic synthesis of the corresponding benzene oxides and of a substituted oxepine, via dihydrobenzene oxide intermediates. A rapid total racemization of the substituted benzene 2,3-oxides was found to have occurred, via their oxepine valence tautomers, in accord with predictions and theoretical calculations. Reduction of a substituted arene oxide to yield a racemic arene hydrate was observed. Arene hydrates have also been synthesised, in enantiopure form, from the corresponding dihydroarene oxide or trans-bromoacetate precursors. Biotransformation of one arene hydrate enantiomer resulted in a toluene-dioxygenase catalysed cis-dihydroxylation to yield a benzene cis-triol metabolite.

The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3.

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 µmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and benzo[b]furans: synthesis of benzo[b]thiophene 2,3-oxide.

Enzymatic cis-dihydroxylation of benzo[b]thiophene, benzo[b]furan and several methyl substituted derivatives was found to occur in both the carbocyclic and heterocyclic rings. Relative and absolute configurations and enantiopurities of the resulting dihydrodiols were determined. Hydrogenation of the alkene bond in carbocyclic cis-dihydrodiols and ring-opening epimerization/reduction reactions of heterocyclic cis/trans-dihydrodiols were also studied. The relatively stable heterocyclic dihydrodiols of benzo[b]thiophene and benzo[b]furan showed a strong preference for the trans configuration in aqueous solutions. The 2,3-dihydrodiol metabolite of benzo[b]thiophene was utilized as a precursor in the chemoenzymatic synthesis of the unstable arene oxide, benzo[b]thiophene 2,3-oxide.

Base-catalyzed dehydration of 3-substituted benzene cis-1,2-dihydrodiols: stabilization of a cyclohexadienide anion intermediate by negative aromatic hyperconjugation.

Evidence that a 1,2-dihydroxycyclohexadienide anion is stabilized by aromatic "negative hyperconjugation" is described. It complements an earlier inference of "positive" hyperconjugative aromaticity for the cyclohexadienyl cation. The anion is a reactive intermediate in the dehydration of benzene cis-1,2-dihydrodiol to phenol. Rate constants for 3-substituted benzene cis-dihydrodiols are correlated by σ(-) values with ρ = 3.2. Solvent isotope effects for the reactions are k(H(2)O)/k(D(2)O) = 1.2-1.8. These measurements are consistent with reaction via a carbanion intermediate or a concerted reaction with a "carbanion-like" transition state. These and other experimental results confirm that the reaction proceeds by a stepwise mechanism, with a change in rate-determining step from proton transfer to the loss of hydroxide ion from the intermediate. Hydrogen isotope exchange accompanying dehydration of the parent benzene cis-1,2-dihydrodiol was not found, and thus, the proton transfer step is subject to internal return. A rate constant of ~10(11) s(-1), corresponding to rotational relaxation of the aqueous solvent, is assigned to loss of hydroxide ion from the intermediate. The rate constant for internal return therefore falls in the range 10(11)-10(12) s(-1). From these limiting values and the measured rate constant for hydroxide-catalyzed dehydration, a pK(a) of 30.8 ± 0.5 was determined for formation of the anion. Although loss of hydroxide ion is hugely exothermic, a concerted reaction is not enforced by the instability of the intermediate. Stabilization by negative hyperconjugation is proposed for 1,2-dihydroxycyclohexadienide and similar anions, and this proposal is supported by additional experimental evidence and by computational results, including evidence for a diatropic ("aromatic") ring current in 3,3-difluorocyclohexadienyl anion.

Structure, stereochemistry and synthesis of enantiopure cyclohexenone cis-diol bacterial metabolites derived from phenols.

Biotransformation of 3-substituted and 2,5-disubstituted phenols, using whole cells of P. putida UV4, yielded cyclohexenone cis-diols as single enantiomers; their structures and absolute configurations have been determined by NMR and ECD spectroscopy, X-ray crystallography, and stereochemical correlation involving a four step chemoenzymatic synthesis from the corresponding cis-dihydrodiol metabolites. An active site model has been proposed, to account for the formation of enantiopure cyclohexenone cis-diols with opposite absolute configurations.

Chemoenzymatic formal synthesis of (-)- and (+)-epibatidine.

The cis-dihydrocatechol, derived from enzymatic cis-dihydroxylation of bromobenzene using the microorganism Pseudomonas putida UV4, was converted into (-)-epibatidine in eleven steps with complete stereocontrol. In addition, an unprecedented palladium-catalysed disproportionation reaction gave the (+)-enantiomer of an advanced key intermediate employed in a previous synthesis of epibatidine.

Chemoenzymatic synthesis of a mixed phosphine-phosphine oxide catalyst and its application to asymmetric allylation of aldehydes and hydrogenation of alkenes.

The chemoenzymatic synthesis of a Lewis basic phosphine-phosphine oxide organocatalyst from a cis-dihydrodiol metabolite of bromobenzene proceeds via a palladium-catalysed carbon-phosphorus bond coupling and a novel room temperature Arbuzov [2,3]-sigmatropic rearrangement of an allylic diphenylphosphinite. Allylation of aromatic aldehydes were catalysed by the Lewis basic organocatalyst giving homoallylic alcohols in up to 57% ee. This compound also functioned as a ligand for rhodium-catalysed asymmetric hydrogenation of acetamidoacrylate giving reduction products with ee values of up to 84%.

Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes.

Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring.

Hyperaromatic stabilization of arenium ions: a remarkable cis stereoselectivity of nucleophilic trapping of ß-hydroxyarenium ions by water.

Cis- and trans-1,2-dihydrodiol isomers of benzene undergo acid-catalyzed dehydration to form phenol. In principle the isomeric substrates react through a common ß-hydroxybenzenium (cyclohexadienyl) carbocation. Notwithstanding, the isomers show a large difference in reactivity, k(cis)/k(trans) = 4500. This difference is reduced to k(cis)/k(trans) = 440 and 50 for the 1,2-dihydrodiols of naphthalene and 9,10-dihydrodiols of phenanthrene, respectively, and to 6.9 for the dihydrodiols of the nonaromatic 7,8-double bond of acenaphthylene. Because the difference in stabilities of cis- and trans-dihydrodiols should be no more than 2-3-fold, these results imply a high cis stereoselectivity for nucleophilic trapping of a ß-hydroxyarenium cation by water in the reverse of the carbocation-forming reaction. This is confirmed by studies of the 10-hydroxy-9-phenanthrenium ion generated from aqueous solvolyses of the trans-9,10-bromohydrin derivative of phenanthrene and the monotrichloroacetate ester of the phenanthrene cis-9,10-dihydrodiol. The cis stereoselectivity of forward and reverse reactions is explained by the formation (in the "forward" reaction) of different conformations of carbocation from cis- and trans-dihydrodiol reactants with respectively ß-C-H and ß-C-OH bonds in pseudoaxial positions with respect to the charge center of the carbocation optimal for hyperconjugation. Formation of different conformations is constrained by departure of the (protonated) OH leaving group from a pseudoaxial position. The difference in stability of the carbocations is suggested to stem (a) from the greater hyperconjugative ability of a C-H than a C-OH bond and (b) from enhanced conjugation arising from the stabilizing influence of an aromatic ring in the no-bond resonance structures representing the hyperconjugation (C(6)H(6)OH(+) ↔ C(6)H(5)OH H(+)). This is consistent with an earlier suggestion by Mulliken and a demonstration by Schleyer that the benzenium ion is subject to hyperconjugative aromatic stabilization. It is proposed that, in analogy with the terms homoconjugation and homoaromaticity, arenium ions should be considered as "hyperaromatic".

Acid-catalyzed dehydration of naphthalene-cis-1,2-dihydrodiols: origin of impaired resonance effect of 3-substituents.

Acid-catalyzed dehydrations of substituted naphthalene-cis-1,2-dihydrodiols occur with loss of the 1- or 2-OH group to form 2- and 1-naphthols, respectively. Effects of substituents MeO, Me, H, F, Br, I, and CN at 3-, 6-, and 7-positions of the naphthalene ring are consistent with rate-determining formation of ß-hydroxynaphthalenium ion (carbocation) intermediates. For reaction of the 1-hydroxyl group the 3-substituents are correlated by the Yukawa-Tsuno relationship with ρ = -4.7 and r = 0.25 or by σ(p) constants with ρ = -4.25; for reaction of the 2-hydroxyl group the 3-substituents are correlated by σ(m) constants with ρ = -8.1. The correlations for the 1-hydroxyl imply a surprisingly weak resonance interaction of +M substituents (MeO, Me) with a carbocation reaction center but are consistent with the corresponding correlation for acid-catalyzed dehydration of 3-substituted benzene-cis-1,2-dihydrodiols for which ρ = -6.9 and r = 0.43. Substituents at the 6- and 7-positions of the naphthalene rings by contrast are correlated by σ(+) with ρ = -3.2 for reaction of the 1-hydroxyl group and ρ = -2.7 for reaction of the 2-hydroxyl group. The unimpaired resonance implied by these substituent effects appears to be inconsistent with a previous explanation of the weak resonance of the 3-substituents in terms of imbalance of charge development and/or nonplanarity of the benzenium ring in the transition state. An alternative possibility is that the adjacent hydroxyl group interferes sterically with conjugation of +M substituents. "Hyperaromaticity" of the arenium ion intermediates does not appear to be a factor influencing this behavior.