Microbial Synthesis of (S)- and (R)-Benzoin in Enantioselective Desymmetrization and Deracemization Catalyzed by Aureobasidium pullulans Included in the Blossom Protect™ Agent.

In this study, we examined the Aureobasidium pullulans strains DSM 14940 and DSM 14941 included in the Blossom Protect™ agent to be used in the bioreduction reaction of a symmetrical dicarbonyl compound. Both chiral 2-hydroxy-1,2-diphenylethanone antipodes were obtained with a high enantiomeric purity. Mild conditions (phosphate buffer [pH 7.0, 7.2], 30 °C) were successfully employed in the synthesis of (S)-benzoin using two different methodologies: benzyl desymmetrization and rac-benzoin deracemization. Bioreduction carried out with higher reagent concentrations, lower pH values and prolonged reaction time, and in the presence of additives, enabled enrichment of the reaction mixture with (R)-benzoin. The described procedure is a potentially useful tool in the synthesis of chiral building blocks with a defined configuration in a simple and economical process with a lower environmental impact, enabling one-pot biotransformation.

Supramolecular chirality and crystallization from biocatalytic self-assembly in lipidic cubic mesophases.

Biocatalytic self-assembly in a nanoconfined environment is widely used in nature to construct complex structures that endow special characteristics to life. There is tremendous interest in mimicking such bottom-up processes to fabricate functional materials. In this study, we have investigated a novel biomimetic scaffold based on lipidic cubic mesophases (LCMs), which provide a special nanoconfined environment for biocatalytic self-assembly and subsequent formation of organic crystals. (R)-Benzoin generated in situ from benzaldehyde in a reaction catalyzed by the enzyme benzaldehyde lyase (BAL) exhibits - when confined within LCMs - enhanced chirality compared to (R)-benzoin in solution or (R)-benzoin-doped LCMs. We infer that a metastable state is formed under kinetic control that displays enhanced supramolecular chirality. As they age, these metastable structures can further grow into thermodynamically stable crystals. The biomimetic, nanoconfined environment provided by the LCMs plays a key role in the development of supramolecular chirality and subsequent crystallization.

Biotransformation of benzoin by Sphingomonas sp. LK11 and ameliorative effects on growth of Cucumis sativus.

Plant endophytes play vital role in plant growth promotion as well as in abiotic and biotic stress tolerance. They also mediate biotransformation of complex organic materials to simpler and useful by-product. Therefore, the role of plant endophyte in plant growth promotion and stress tolerance has gained considerable attention in recent days. Sphingomonas sp. LK11 is an important plant endophyte that actively regulates plant growth. However, the biotransformation and stress tolerance potential of Sphingomonas sp. LK11 was yet to be elucidated. Therefore, we studied the biotransformation of benzoin by Sphingomonas sp. LK11. We found that, Sphingomonans sp. LK11 biotransformed benzoin to benzamide. Further application of benzamide to Cucumis sativus led to decrease in agronomic potential of C. sativus as benzamide acts as an abiotic stress agent. However, the application of Sphingomonas sp. LK11 inoculums with benzamide reverted back the agronomic trait of the plants, suggesting the role of Sphingomonas sp. LK11 in biotransformation and abiotic stress tolerance in plants.

Ligand-Based Drug Design: Synthesis and Biological Evaluation of Substituted Benzoin Derivatives as Potential Antitumor Agents.

BACKGROUND: Phosphoinositide 3-kinase α (PI3Kα) has emerged as a promising target for anticancer drug design. OBJECTIVES: Target compounds were designed to investigate the effect of the p-OCH3 motifs on ligand/PI3Kα complex interaction and antiproliferative activity. METHODS: Synthesis of the proposed compounds, biological examination tests against human colon adenocarcinoma (HCT-116), breast adenocarcinoma (MCF-7), and breast carcinoma (T47D) cell lines, along with Glide docking studies. RESULTS: A series of 1,2-bis(4-methoxyphenyl)-2-oxoethyl benzoates was synthesized and characterized by means of FT-IR, 1H and 13C NMR, and by elemental analysis. Biological investigation demonstrated that the newly synthesized compounds exhibit antiproliferative activity in human colon adenocarcinoma (HCT-116), breast adenocarcinoma (MCF-7), and breast carcinoma (T47D) cell lines possibly via inhibition of PI3Kα and estrogen receptor alpha (ERα). Additionally, results revealed that these compounds exert selective inhibitory activity, induce apoptosis, and suppress VEGF production. Compound 3c exhibited promising antiproliferative activity in HCT-116 interrogating that hydrogen bond-acceptor mediates ligand/PI3Kα complex formation on m- position. Compounds 3e and 3i displayed high inhibitory activity in MCF-7 and T47D implying a wide cleft discloses the o-attachment. Furthermore, compound 3g exerted selective inhibitory activity against T47D. Glide docking studies against PI3Kα and ERα demonstrated that the series accommodate binding to PI3Kα and/or ERα. CONCLUSION: The series exhibited a potential antitumor activity in human carcinoma cell lines encoding PI3Kα and/or ERα.

Intramolecular Benzoin Reaction Catalyzed by Benzaldehyde Lyase from Pseudomonas Fluorescens Biovar I.

Intramolecular benzoin reactions catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I (BAL) are reported. The structure of the substrates envisaged for this reaction consists of two benzaldehyde derivatives linked by an alkyl chain. The structural requirements needed to achieve the intramolecular carbon-carbon bond reaction catalyzed by BAL were established. Thus, a linker consisting of a linear alkyl chain of three carbon atoms connected through ether-type bonds to the 2 and 2' positions of two benzaldehyde moieties, which could be substituted with either Cl, Br, or OCH3 at either the 3 and 3' or 5 and 5' positions, were suitable substrates for BAL. Reactions with 61-84 % yields of the intramolecular product and ee values between 64 and 98 %, were achieved.

A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil.

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

Biphasic mini-reactor for characterization of biocatalyst performance.

Biphasic reaction media are extending the scope of technical biocatalysis. Thorough investigation of the factors affecting catalyst performance under these conditions is of key importance for the successful implementation of catalytic processes. Here, we present a reactor setup suitable for comprehensive systematic characterization and optimization of biocatalyzed reactions in biphasic systems with distinct phases. It is distinguished by small volumes allowing reproducible experimentation with minimum amounts of solvent and catalyst. The interfacial area is kept constant and independent stirring of both phases is allowed in order to minimize superimposing effects. Evaporation of low-volatile organic solvents is prevented by use of airtight construction. The broad applicability of this mini-reactor is demonstrated with regard to determination of mass transfer, enzyme productivity, and enzyme stability in both batch and continuous mode.

Artificial enzymes with thiazolium and imidazolium coenzyme mimics.

Hydrophobic thiazolium and imidazolium coenzyme mimics in the presence of modified-polyethylenimine enzyme mimics catalyze the benzoin condensation 2300-3300 times faster than the coenzyme mimics alone. Polycationic enzyme mimics provide not only a hydrophobic binding domain for coenzyme and substrate, but also electrostatic stabilization of anionic species that arise along the reaction pathway of the benzoin condensation.

Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester.

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

Asymmetric reduction of benzil to (S)-benzoin with Penicillium claviforme IAM 7294 in a liquid-liquid interface bioreactor (L-L IBR).

The asymmetric reduction of benzyl to (S)-benzoin with Penicillium claviforme IAM 7294 was applied to a liquid-liquid interface bioreactor (L-L IBR) using a unique polymeric material, ballooned microsphere (MS). The L-L IBR showed superior performance, as compared with suspension, organic-aqueous two-liquid-phase, and solid-liquid interface bioreactor (S-L IBR) systems, affording 14.4 g/l-organic phase of (S)-benzoin (99.0% ee).

Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

Antiplasmodial activity of [(aryl)arylsulfanylmethyl]Pyridine.

A series of [(aryl)arylsufanylmethyl]pyridines (AASMP) have been synthesized. These compounds inhibited hemozoin formation, formed complexes (K(D) = 12 to 20 muM) with free heme (ferriprotoporphyrin IX) at a pH close to the pH of the parasite food vacuole, and exhibited antimalarial activity in vitro. The inhibition of hemozoin formation may develop oxidative stress in Plasmodium falciparum due to the accumulation of free heme. Interestingly, AASMP developed oxidative stress in the parasite, as evident from the decreased level of glutathione and increased formation of lipid peroxide, H(2)O(2), and hydroxyl radical (.OH) in P. falciparum. AASMP also caused mitochondrial dysfunction by decreasing mitochondrial potential (DeltaPsim) in malaria parasite, as measured by both flow cytometry and fluorescence microscopy. Furthermore, the generation of .OH may be mainly responsible for the antimalarial effect of AASMP since .OH scavengers such as mannitol, as well as spin trap alpha-phenyl-n-tertbutylnitrone, significantly protected P. falciparum from AASMP-mediated growth inhibition. Cytotoxicity testing of the active compounds showed selective activity against malaria parasite with selectivity indices greater than 100. AASMP also exhibited profound antimalarial activity in vivo against chloroquine resistant P. yoelii. Thus, AASMP represents a novel class of antimalarial.

Enzyme-purification and catalytic transformations in a microstructured PASSflow reactor using a new tyrosine-based Ni-NTA linker system attached to a polyvinylpyrrolidinone-based matrix.

The synthesis of a Ni-nitrilotriacetic acid (Ni-NTA) attached via a new tyrosine-based linker matrix on monolithic crosslinked poly(vinyl benzyl chloride)/poly(vinylpyrrolidinone) is described. This matrix is incorporated inside a microstructured PASSflow reactor which was used for automatic purification and immobilisation of His(6)-tagged proteins. These could be used as stable and highly active biocatalysts for the synthesis of (R)-benzoin (6), (R)-2-hydroxy-1-phenylpropan-1-one (7) and 6-O-acetyl-D-glucal (17) in a flow-through mode.

Deoxybenzoins are novel potent selective estrogen receptor modulators.

Deoxybenzoins are plant compounds with similar structure to isoflavones. In this study, we evaluated the ability of two synthesized deoxybenzoins (compound 1 and compound 2) (a) to influence the activity of the estrogen receptor subtypes ERalpha and ERbeta in HeLa cells co-transfected with an estrogen response element-driven luciferase reporter gene and ERalpha- or ERbeta-expression vectors, (b) to modulate the IGFBP-3 and pS2 protein in MCF-7 breast cancer cells, (c) to induce mineralization of KS483 osteoblasts and (d) to affect the cell viability of endometrial (Ishikawa) and breast (MCF-7, MDA-MB-231) cancer cells. Docking and binding energy calculations were performed using the mixed Monte Carlo/Low Mode search method (Macromodel 6.5). Compound 1 displayed significant estrogenic activity via ERbeta but no activity via ERalpha. Compound 2 was an estrogen-agonist via ERalpha and antagonist via ERbeta. Both compounds increased, like the pure antiestrogen ICI182780, the IGFBP-3 levels. Compound 2 induced, like 17beta-estradiol, significant mineralization in osteoblasts. The cell viability of Ishikawa cells was unchanged in the presence of either compound. Compound 1 increased MCF-7 cell viability consistently with an increase in pS2 levels, whereas compound 2 inhibited the cell viability. Molecular modeling confirmed the agonistic or antagonistic behaviour of compound 2 via ER subtypes. Compound 2, being an agonist in osteoblasts, an antagonist in breast cancer cells, with no estrogenic effects in endometrial cancer cells, makes it a potential selective estrogen receptor modulator and a choice for hormone replacement therapy.

Asymmetric reduction of benzil to (S)-benzoin with whole cells of Bacillus cereus.

Benzil (1) was selectively reduced to (S)-benzoin (2) in the presence of a wild-type Bacillus cereus Tim-r01. A 92% yield of 2 with 94% enantiomeric excess ratio was attained in phosphate-buffered saline (PBS) (pH 7.5) by using glucose as a nutrient at 37 degrees C for 12 h. Compound 2 was not reduced further to hydrobenzoin (3) at all. The reduction activity differed greatly depending on the strain of B. cereus. Under these conditions the B. cereus strains IFO3001, IFO15305, IAM1110, IAM1229, IAM1656, and IAM1729 gave 2 in yields ranging from 23 to 46% and the configuration of 2 was (S)-form (7 to 86% ee).

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. II. Comparison of chiral recognition properties with crude ovomucoid.

Chiral stationary phases based on ovoglycoprotein from chicken egg whites (OGCHI) and crude ovomucoid from chicken egg whites (OMCHI) were compared with regard to the bound amounts of OGCHI and chiral recognition abilities. Crude OMCHI included 11% OGCHI, by weight. Since pure OMCHI had no appreciable chiral recognition ability, the chiral recognition ability of crude OMCHI originated from OGCHI, which was present in crude OMCHI preparations as an impurity. However, a chiral stationary phase based on crude OMCHI showed good chiral recognition ability, despite the 11% OGCHI content in crude OMCHI preparations. When crude OMCHI was reacted with N,N'-disuccinimidylcarbonate (DSC)-activated aminopropyl-silica gels, the ratio of bound OGCHI to that of totally bound protein was 0.23. These results reveal that the good chiral recognition ability of a stationary phase based on crude OMCHI is due to OGCHI being preferentially bound to DSC-activated aminopropyl-silica gels rather than the OMCHI. In addition, OMCHI did not contribute to the enantioselectivity of the solute at all and made little contribution to the retention characteristics.