Dioxygenase-catalysed oxidation of disubstituted benzene substrates: benzylic monohydroxylation versus aryl cis-dihydroxylation and the meta effect

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric substituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.     

Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.     

Dioxygenase-catalysed oxidation of monosubstituted thiophenes: sulfoxidation versus dihydrodiol formation

Toluene dioxygenase (TDO)-catalysed sulfoxidation, using Pseudomonas putida UV4, was observed for the thiophene substrates 1A-1N. The unstable thiophene oxide metabolites, 6A-6G, 6K-6N, spontaneously dimerised yielding the corresponding racemic disulfoxide cycloadducts 7A-7G, 7K-7N. Dimeric or crossed [4 + 2] cycloaddition products, derived from the thiophene oxide intermediates 6A and 6D or 6B and 6D, were found when mixtures of thiophene substrates 1A and 1D or 1B and 1D were biotransformed. The thiophene sulfoxide metabolite 6B was also trapped as cycloadducts 17 or 18 using stable dienophiles. Preferential dioxygenase-catalysed oxidation of the substituent on the thiophene ring, including exocyclic sulfoxidation (1H-1J) and cis-dihydroxylation of a phenyl substituent (1G and 1N), was also observed. An enzyme-catalysed deoxygenation of a sulfoxide in P. putida UV4 was noticed when racemic disulfoxide cyclo-adducts 7A, 7B and 7K were converted to the corresponding enantioenriched monosulfoxides 8A, 8B and 8K via a kinetic resolution process. The parent thiophene 1A and the 3-substituted thiophenes 1K-1N were also found to undergo ring dihydroxylation yielding the cis/trans-dihydrodiol metabolites 9A and 9K-9N. Evidence is provided for a dehydrogenase-catalysed desaturation of a heterocyclic dihydrodiol (9Kcis/9Ktrans) to yield the corresponding 2,3-dihydroxythiophene (24) as its preferred thiolactone tautomer (23). A simple model to allow prediction of the structure of metabolites, formed from TDO-catalysed bacterial oxidation of thiophene substrates 1, is presented.     

Regioselectivity and stereoselectivity of dioxygenase catalysed cis-dihydroxylation of mono- and tri-cyclic azaarene substrates

cis-Dihydrodiol metabolites were obtained from dioxygenase-catalysed asymmetric dihydroxylations of five monocyclic (azabiphenyl) and four tricyclic (azaphenanthrene) azaarene substrates. Enantiopurity values and absolute configuration assignments were determined using a combination of stereochemical correlation, X-ray crystallography and spectroscopy methods. The degree of regioselectivity found during cis-dihydroxylation of monocyclic azaarenes (2,3 bond > 3,4 bond) and of tricyclic azaarenes (bay region > non-bay region bonds) was dependent on the type of dioxygenase used. The cis-dihydrodiol metabolite from an azaarene (3-phenylpyridine) was utilised in the chemoenzymatic synthesis of the corresponding trans-dihydrodiol.     

Chemoenzymatic synthesis of trans-dihydrodiol derivatives of monosubstituted benzenes from the corresponding cis-dihydrodiol isomers

Enantiopure trans-dihydrodiols have been obtained by a chemoenzymatic synthesis from the corresponding cis-dihydrodiol metabolites, obtained by dioxygenase-catalysed arene cis-dihydroxylation at the 2,3-bond of monosubstituted benzene substrates. This generally applicable, seven-step synthetic route to trans-dihydrodiols involves a regioselective hydrogenation and a Mitsunobu inversion of configuration at C-2, followed by benzylic bromination and dehydrobromination steps. The method has also been extended to the synthesis of both enantiomers of the trans-dihydrodiol derivatives of toluene, through substitution of a vinyl bromine atom of the corresponding trans-dihydrodiol enantiomers derived from bromobenzene. Through incorporation of hydrogenolysis and diMTPA ester diastereoisomer resolution steps into the synthetic route, both trans-dihydrodiol enantiomers of monohalobenzenes were obtained from the cis-dihydrodiols of 4-haloiodobenzenes.     

Chemoenzymatic synthesis of the trans-dihydrodiol isomers of monosubstituted benzenes via anti-benzene dioxides

Enantiopure cis-2,3-dihydrodiols, available from dioxygenase-catalysed cis-dihydroxylation of monosubstituted benzene substrates, have been used as synthetic precursors of the corresponding trans-3,4-dihydrodiols. The six-step chemoenzymatic route from cis-dihydrodiol precursors, involving acetonide, tetraol, dibromodiacetate and diepoxide intermediates, and substitution of vinyl bromide and iodide atoms, has been used in the synthesis of ten trans-dihydrododiol derivatives of substituted benzenes. The general applicability of the method has been demonstrated by its use in the synthesis of both enantiomers of the trans-1,2-and 3,4-dihydrodiol derivatives of toluene.     

Mechanism of the oxidation of sulfides by dioxiranes. 1. Intermediacy of a 10-S-4 hypervalent sulfur adduct

Earlier studies established that dimethyldioxirane (1a) reacts with sulfides 2 in two consecutive concerted electrophilic oxygen-transfer steps to give first sulfoxides 3 and then sulfones 4. The same sequential electrophilic oxidation model was assumed for the reaction of sulfides 2 with the strongly electrophilic methyl(trifluoromethyl)dioxirane (1b). In this paper we report on a systematic and general study on the mechanism of the reaction of simple sulfides 2 with DMDO (1a) and TFDO (1b) which provides clear evidence for the involvement of hypervalent sulfur species in the oxidation process. In the oxidation of sulfides 2a-c, diphenyl sulfide (2d), para-substituted aryl methyl sulfides 2e-i, and phenothiazine 2k with 1b, the major product was the corresponding sulfone 4, even when a 10-fold excess of sulfide relative to 1b was used. The sulfone:sulfoxide 4:3 ratio depends among other factors on the dioxirane 1a or 1b used, the sulfide substitution pattern, the polar, protic, or aprotic character of the solvent, and the temperature. The influence of these factors and also deuterium and (18)O tracer experiments performed allow a general mechanism to be depicted for these oxidations in which the key step is the reversible cyclization of a zwitterionic intermediate, 6, to form a hypervalent sulfur species, 7. The classical sequential mechanism which establishes that sulfides are oxidized first to sulfides and then to sulfones can be enclosed in our general picture of the process and represents just those particular cases in which the zwitterionic intermediate 6 decomposes prior to undergoing ring closure to afford the hypervalent sulfurane intermediate 7.     

Iron(III) [bond] Salen complexes as enzyme models: mechanistic study of oxo(salen)iron complexes oxygenation of organic sulfides

The oxidation of a series of para-substituted phenyl methyl sulfides was carried out with several oxo(salen)iron (salen = N,N'-bis(salicylidine)ethylenediaminato) complexes in acetonitrile. The oxo complex [O=Fe(IV)(salen)](*+), generated from an iron(III) [bond] salen complex and iodosylbenzene, effectively oxidizes the organic sulfides to the corresponding sulfoxides. The formation of [O [double bond] Fe(IV)(salen)](*+) as the active oxidant is supported by resonance Raman studies. The kinetic data indicate that the reaction is first-order in the oxidant and fractional-order with respect to sulfide. The observed saturation kinetics of the reaction and spectral data indicate that the substrate binds to the oxidant before the rate-controlling step. The rate constant (k) values for the product formation step determined using Michaelis-Menten kinetics correlate well with Hammett sigma constants, giving reaction constant (rho) values in the range of -0.65 to -1.54 for different oxo(salen)iron complexes. The log k values observed in the oxidation of each aryl methyl sulfide by substituted oxo(salen)iron complexes also correlate with Hammett sigma constants, giving positive rho values. The substituent effect, UV-vis absorption, and EPR spectral studies indicate oxygen atom transfer from the oxidant to the substrate in the rate-determining step.     

Stereoselective reductase-catalysed deoxygenation of sulfoxides in aerobic and anaerobic bacteria

Direct and indirect evidence, of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found. The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4. Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains. A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.     

Alloxazine-cyclodextrin conjugates for organocatalytic enantioselective sulfoxidations

Four structurally different alloxazine-cyclodextrin conjugates were prepared and tested as catalysts for the enantioselective oxidation of prochiral sulfides to sulfoxides by hydrogen peroxide in aqueous solutions. The alloxazinium unit was appended to the primary face of α- and β-cyclodextrins via a linker with variable length. A series of sulfides was used as substrates: n-alkyl methyl sulfides (n-alkyl = hexyl, octyl, decyl, dodecyl), cyclohexyl methyl sulfide, tert-butyl methyl sulfide, benzyl methyl sulfide and thioanisol. α-Cyclodextrin conjugate having alloxazinium unit attached via a short linker proved to be a suitable catalyst for oxidations of n-alkyl methyl sulfides, displaying conversions up to 98% and enantioselectivities up to 77% ee. β-Cyclodextrin conjugates were optimal catalysts for the oxidation of sulfides carrying bulkier substituents; e.g. tert-butyl methyl sulfide was oxidized with quantitative conversion and 91% ee. Low loadings (0.3-5 mol%) of the catalysts were used. No overoxidation to sulfones was observed in this study.     

Asymmetric sulfoxidation of a β-carbonyl sulfide series by chloroperoxidase

The chloroperoxidase (CPO)-catalyzed oxidation of a series of β-carbonyl sulfides to sulfoxides has been studied at room temperature in aqueous citrate buffer. For dialkyl β-carbonyl sulfides, the products with methyl and ethyl substituents are obtained in ca. 100% yield. However when the alkyl group is n-propyl or i-propyl the yield drops dramatically (25%). An aryl sulfide derivative afforded product in very low yield (4%), but when the phenyl group bears a carbonyl, and the sulfur substituents are methyl or ethyl, the oxidation occurs with high yields (91–95%). Steric control of the sulfoxidation reaction is also confirmed with cyclohexanone derivatives, where a low product yield is observed even at high enzyme concentrations. Noteworthy are the yields obtained with cyclopentanone sulfide (65%) and an unexpected quantitative yield obtained with the γ-butyrolactone sulfide.     

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.     

Probing the scope of the sulfoxidation activity of vanadium bromoperoxidase from Ascophyllum nodosum

It has been shown previously that the vanadium bromoperoxidase from Ascophyllum nodosum mediates the production of (R)-methyl phenyl sulfoxide with 91% enantiomeric excess from the corresponding sulfide in the presence of hydrogen peroxide. Investigation of the sulfoxidation activity of this enzyme shows that activating substituents at the para-position of the aromatic ring of methyl phenyl sulfide positively influence the selectivity of the reaction, whereas strongly electron-withdrawing groups cancel the catalyzed sulfoxidation reaction. The first evidence is presented that the vanadium bromoperoxidase catalyzes the sulfoxidation of racemic non-aromatic cyclic thioethers with high kinetic resolution.     

Asymmetric oxidation of sulfides catalyzed by chiral (salen)Mn(III) complexes with a pyrrolidine backbone

Catalytic properties of a series of chiral (pyrrolidine salen)Mn(III) complexes for asymmetric oxidation of aryl methyl sulfides were evaluated. Moderate activity, good chemical selectivity and low enantioselectivity were attained with iodosylbenzene as a terminal oxidant. Enantioselectivity of sulfide oxidation was affected slightly by polar solvent and the sulfoxidation carried out in THF for thioanisole and in CH₃CO₂Et for electron‐deficient sulfides gave better enatioselctivities. The addition of the donor ligand PPNO (4‐phenylpyridine N‐oxide) or MNO (trimethylamine N‐oxide) only has a minor positive effect on the enantioselectivity. Also explored was the steric effect of the N_aza‐substituent in the backbone of (pyrrolidine salen)Mn(III) complexes on the enantioselectivity of sulfide oxidation. The sulfides' access pathway is discussed based on the catalytic results. Copyright © 2006 John Wiley & Sons, Ltd.     

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.     

Molecular addition compounds. 17. Borane and chloroborane adducts with organic sulfides for hydroboration

The following sulfides have been examined as borane carriers in comparison with dimethyl sulfide and 1,4-oxathiane: tert-butyl methyl sulfide, isoamyl methyl sulfide, ethyl isoamyl sulfide, tert-butyl isoamyl sulfide, diisoamyl sulfide, tetrahydrothiophene, tetrahydro-thiopyran, thioanisole, 3-ethylthiotetrahydrofuran, bis(3-tetrahydrofuryl) sulfide, and bis(2-methoxyethyl) sulfide. Their complexing ability toward borane increases in the following order: thioanisole < ether-sulfides < dialkyl sulfides < dimethyl sulfide. Borane adducts of the sulfides are liquids above 0 degrees C. The thioanisole adduct loses diborane at room temperature. The reactivity of the adducts toward 1-octene increases in the reversed order of the complexing ability of the sulfides. Diisoamyl sulfide has a mild, ethereal, agreeable aroma, its synthesis is economical and the borane adduct, 4.2 M in BH3, is stable over prolonged periods at room temperature. The sulfide can be recovered from hydroboration-oxidation products by distillation. Consequently, diisoamyl sulfide is a new promising borane carrier. Bis(2-methoxyethyl) sulfide, easily synthesized from the low cost thiodiethanol, is three times more soluble in water than 1,4-oxathiane. Its borane adduct is 6.0 M in BH3 and can substitute for more expensive borane-1,4-oxathiane in hydroboration reactions. Applications of these new borane adducts in the synthesis of mono- and dichloroborane adducts was also studied. The equilibrium ratios observed for the new chloroborane adducts were similar to that observed for dimethyl sulfide adducts. However, the hydroboration of 1-octene with these new chloroborane adducts are much faster than the corresponding adducts of dimethyl sulfide, which are currently used extensively.     

Copper-catalyzed asymmetric oxidation of sulfides

Copper-catalyzed asymmetric sulfoxidation of aryl benzyl and aryl alkyl sulfides, using aqueous hydrogen peroxide as the oxidant, has been investigated. A relationship between the steric effects of the sulfide substituents and the enantioselectivity of the oxidation has been observed, with up to 93% ee for 2-naphthylmethyl phenyl sulfoxide, in modest yield in this instance (up to 30%). The influence of variation of solvent and ligand structure was examined, and the optimized conditions were then used to oxidize a number of aryl alkyl and aryl benzyl sulfides, producing sulfoxides in excellent yields in most cases (up to 92%), and good enantiopurities in certain cases (up to 84% ee).     

4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides

Recombinant 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB has been tested as a catalyst in sulfoxidation reactions on a set of aromatic sulfides. With a few exceptions, excellent enantioselectivities in the synthesis of chiral phenyl and benzyl sulfoxides were achieved. The bacterial Baeyer–Villiger monooxygenase was also shown to accept racemic sulfoxides, a prochiral diketone and an organoboron compound as substrates. This study demonstrates the great biocatalytic potential of this novel oxidative enzyme.     

New designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2) in rat urine using gas chromatography/mass spectrometry (GC/MS) after enzymatic cleavage of conjugates, liquid-liquid extraction and derivatization. The structures of 14 metabolites were assigned tentatively by detailed interpretation of their mass spectra. Identification of these metabolites indicated that 2C-T-2 was metabolized by sulfoxidation followed by N-acetylation and either hydroxylation of the S-ethyl side chain or demethylation of one methoxy group, O-demethylation of the parent compound followed by N-acetylation and sulfoxidation, deamination followed by reduction to the corresponding alcohol followed by partial glucuronidation and/or sulfation or by oxidation to the corresponding acid followed either by partial glucuronidation or by degradation to the corresponding benzoic acid derivative followed by partial glucuronidation. Furthermore, 2C-T-2 was metabolized by N-acetylation of the parent compound followed either by O-demethylation and sulfoxidation or by S-dealkylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-2 in rat urine, which corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-2 in human urine.     

Asymmetric oxidation of sulfides with H2O2 catalyzed by titanium complexes of Schiff bases bearing a dicumenyl salicylidenyl unit

The sterically hindered Schiff bases (L³–L⁵), prepared from 3,5‐dicumenyl salicylaldehyde and chiral amino alcohols, were used in combination with Ti(OiPr)₄ for asymmetric oxidation of aryl methyl sulfides with H₂O₂ as terminal oxidant. Among the ligands L³–L⁵, L⁴ with a tert‐butyl group in the chiral carbon of the amino alcohol moiety gave the best result with 89% yield and 73% ee for the sulfoxidation of thioanisole under optimal conditions [with 1 mol% of Ti(OiPr)₄ in a molar ratio of 100:1:1.2:120 for sulfide:Ti(OiPr)₄:ligand:H₂O₂ in CH₂Cl₂ at 0 °C for 3 h]. The reaction afforded good yield (84%) with a moderate enantioselectivity (62% ee) even with a lower catalyst loading from 1.0 to 0.5 mol%. The oxidations of methyl 4‐bromophenyl sulfide and methyl 4‐methoxyphenyl sulfide with H₂O₂ catalyzed by the Ti(OiPr)₄–L⁴ system gave 79–84% yields and 54–59% ee of the corresponding sulfoxides in CH₂Cl₂ at 20 °C. The chiral induction capability of the cumenyl‐modified sterically hindered Schiff bases for sulfoxidation was compared with the conventional Schiff bases bearing tert‐butyl groups at the 3,5‐positions of the salicylidenyl unit. Copyright © 2011 John Wiley & Sons, Ltd.