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.

Production of a chiral alcohol, 1-(3,4-dihydroxyphenyl) ethanol, by mushroom tyrosinase.

1-(3,4-Dihydroxyphenyl) ethanol was produced biocatalytically for the first time using mushroom tyrosinase. 4-Ethylphenol at 1 mM was consumed over 12 min giving 0.23 mM 4-ethylcatechol and 0.36 mM (R/S)-1-(3,4-dihydroxyphenyl) ethanol (ee 0.5 %). Mushroom tyrosinase consumed 4-ethylphenol at 6.7 µmol min(-1) mg protein(-1) while the rates of formation of 4-ethylcatechol and 1-(3,4-dihydroxyphenyl) ethanol were 1.1 and 1.9 µmol min(-1) mg protein(-1). Addition of the ascorbic acid, as a reducing agent to biotransformation reactions, increased 4-ethylcatechol formation by 340 %. However, accumulation of 1-(3,4-dihydroxyphenyl) ethanol was not observed in the presence of ascorbic acid. While the 1-(3,4-dihydroxyphenyl) ethanol was racemic, it is the first chiral product produced by tyrosinase starting from a non-chiral substrate.

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.

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase.

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 µmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1-2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5-20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.

Unnatural polyketide analogues selectively target the HER signaling pathway in human breast cancer cells.

Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin-3-galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells (IC(50)>100 microM). Specifically, 4-trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC(50)<200 nM) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2 e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogues might prove to be useful drug candidates for potential breast cancer therapy.

Expanding the Scope of Biocatalysis: Oxidative Biotransformations on Solid-Supported Substrates.

Oxidative biocatalytic reactions were performed on solid-supported substrates, thus expanding the repertoire of biotransformations that can be carried out on the solid phase. Various phenylacetic and benzoic acid analogs were attached to controlled pore glass beads via an enzyme-cleavable linker. Reactions catalyzed by peroxidases (soybean and chloro), tyrosinase, and alcohol oxidase/dehydrogenase gave a range of products, including oligophenols, halogenated aromatics, catechols, and aryl aldehydes. The resulting products were recovered following cleavage from the beads using α-chymotrypsin to selectively hydrolyze a chemically non-labile amide linkage. Controlled pore glass (CPG) modified with a polyethylene glycol (PEG) linker afforded substantially higher product yields than non-PEGylated CPG or non-swellable polymeric resins. This work represents the first attempt to combine solid-phase oxidative biotransformations with subsequent protease-catalyzed cleavage, and serves to further expand the use of biocatalysis in synthetic and medicinal chemistry.

Lewis super-acid catalyzed cyclizations: a new route to fragrance compounds.

This review deals with the application of Lewis super acids such as Al(III), In(III), and Sn(IV) triflates and triflimidates as catalysts in the synthesis of fragrance materials. Novel catalytic reactions involving C-C and C-heteroatom bond-forming reactions, as well as cycloisomerization processes are presented. In particular, Sn(IV) and Al(III) triflates were employed as catalysts in the selective cyclization of unsaturated alcohols to cyclic ethers, as well as in the cyclization of unsaturated carboxylic acids to lactones. The addition of thiols and thioacids to non-activated olefins, both in intra- and intermolecular versions, was efficiently catalyzed by In(III) derivatives. Sn(IV) Triflimidates catalyzed the cycloisomerization of highly substituted 1,6-dienes to gem-dimethyl-substituted cyclohexanes bearing an isopropylidene substituent. The hydroformylation of these unsaturated substrates, catalyzed by a Rh(I) complex with a bulky phosphite ligand, selectively afforded the corresponding linear aldehydes. The olfactory evaluation of selected heterocycles, carbocycles, and aldehydes synthesized is also discussed.

Regioselective indium(III) trifluoromethanesulfonate-catalyzed hydrothiolation of non-activated olefins.

Indium(III) trifluoromethanesulfonate was found to be an excellent catalyst for the highly regioselective intra- and intermolecular addition of thiols to non-activated olefins and could be recycled and reused without loss of activity.

Catalytic formation of C-O bonds by alkene activation: Lewis acid-cycloisomerisation of olefinic alcohols.

Tin(IV) trifluoromethanesulfonate has been found to be an excellent catalyst for the cycloisomerisation of non-activated and differently substituted olefinic alcohols to cyclic ethers.

Aluminium(III) trifluoromethanesulfonate as an efficient catalyst for the intramolecular hydroalkoxylation of unactivated olefins: experimental and theoretical approaches.

The Al(OTf)(3)-catalyzed cycloisomerization of unactivated unsaturated alcohols was studied from experimental and theoretical points of view. A series of cyclic ethers was obtained in excellent yields and regioselectivities. This catalyst system provides one of the most straightforward routes to cyclic ethers with Markovnikov-type regioselectivity under mild conditions. Theoretical and NMR studies were carried out in order to better determine the mechanism of this reaction. The NMR studies were in agreement with preferential complexation of Al(OTf)(3) to the oxygen atom of the unsaturated alcohol, but did not exclude complexation to the double bond of the alcohol. Theoretical calculations indicated strong acidification of the hydroxyl proton when Al(OTf)(3) was complexed to the alcohol oxygen atom. A plausible catalytic cycle for the Al(OTf)(3)-catalyzed intramolecular hydroalkoxylation of unactivated olefins is proposed.