Heterofunctional Magnetic Metal-Chelate-Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity.

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor-made magnetic chelate-epoxy supports. In order to selectively adsorb and then covalently immobilize the poly-His-tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co(2+)-chelate groups (38 µmol Co(2+)/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine-tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free-enzyme-catalyzed reaction. The enantiomeric excess (ee) of (R)-benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles.

Carboligation reactions mediated by benzoylformate decarboxylase immobilized on a magnetic solid support.

In this study, magnetic nanoparticles (Fe3 O4 , magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating-sample magnetometer (VSM) methods for purification and immobilization of the histidine-tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS-tagged BFD by SDS-page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions.

Benzaldehyde lyase catalyzed enantioselective self and cross condensation reactions of acetaldehyde derivatives.

Flexible protected 1,3,4-trihydroxy-2-butanone is synthesized in high enantiomeric excesses by using asymmetric homo- and cross- acyloin coupling of aliphatic aldehydes catalyzed by benzaldehyde lyase.

Enantioselective production of benzoin from benzoin acetate via kinetic resolution and deracemization using Rhizopus oryzae.

In this study, the production of enantiopure benzoin from rac-benzoin acetate was achieved by lipase catalyzed kinetic resolution combined with deracemization using Rhizopus oryzae (CBS111718). The growth cells were pretreated with 20 kHz and 30 kHz ultrasound irradiation and mechanical homogenization. Approximately 100% conversion and 96% enantiomeric excess of the product (S-benzoin) were obtained by applying 20 kHz ultrasound irradiation at pH 6. The deracemization process involves new and important processes that allow for the transformation of a racemate into a single stereoisomeric product in 100% theoretical yields. Moreover, the application of ultrasound increases the conversion rate by reducing mass transfer limitation.

Direct enantioselective aldol reactions catalyzed by a proline-thiourea host-guest complex.

Proline-thiourea host-guest complex-catalyzed direct enantioselective aldol reactions have been developed, in which the catalytic activities were evaluated in the direct asymmetric aldol reactions of various aromatic aldehydes and cyclohexanone.

Catalytic intermolecular aldehyde-ketone coupling via acyl phosphonates.

The first catalytic intermolecular aldehyde-ketone coupling via acyl phosphonate is reported. Acyl phosphonates are potent acyl anion precursors, which generate acyl anion equivalents under the influence of cyanide anion via phosphonate-phosphate rearrangement. These anions readily react with activated ketones to form acyloin type coupling in 41-95% yields.

Carboligation reactions with benzaldehyde lyase immobilized on superparamagnetic solid support.

Histidine-tagged recombinant benzaldehyde lyase (BAL, EC 4.1.2.38) was efficiently immobilized to surface-modified magnetic particles with affinity ligand binding. In addition to conventional benzoin condensation reactions, two important representative BAL-catalyzed carboligation reactions, were also performed with this magnetically responsive biocatalyst. The results obtained from the carboligation reactions that were performed with this simple and convenient heterogenous biocatalyst were comparable to that of free-enzyme-catalyzed reactions.

Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies.

In the light of known HDAC inhibitors, 33 carboxylic acid derivatives were tested to understand the structural requirements for HDAC inhibition activity. Several modifications were applied to develop the structure-activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor-enzyme complexes. Of these compounds, caffeic acid derivatives such as chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, which is a well-known HDAC inhibitor.

Darzens reaction of acyl phosphonates with alpha-bromo ketones: selective synthesis of cis- and trans-epoxyphosphonates.

Acyl phosphonates with alpha-halo ketones in the presence of bases at room temperature afford cis- and trans-epoxyphosphonates in good chemical yields and high selectivities using different bases. The diastereoselectivity of this reaction is easily controlled by changing the base. Changing the base from Cs2CO3 to DBU changed the diastereomeric ratio (trans/cis) from 3/2 to 9/1. Moreover, the treatment of the trans isomer with DBU showed a complete conversion to the corresponding cis isomer.

A new and efficient chemoenzymatic route to both enantiomers of 4-hydroxycyclohex-2-en-1-one.

[reaction: see text] A chemoenzymatic synthesis of both enantiomers of the pharmacologically interesting 4-hydroxycyclohex-2-en-1-one in three steps starting from 3-methoxycyclohex-2-en-1-one is described. Manganese(III) acetate-mediated acetoxylation followed by enzyme-mediated hydrolysis of alpha-acetoxy enone affords acetoxy enone 3 and hydroxy enone 4 with high enantiomeric excesses and in good yields. The reduction of the acetoxy and hydroxy enones furnished both enantiomers of 4-hydroxycyclohex-2-en-1-one in high enantiomeric excess.

Benzaldehyde lyase-catalyzed enantioselective carboligation of aromatic aldehydes with mono- and dimethoxy acetaldehyde.

[reaction: see text] Benzaldehyde lyase from the Pseudomonas fluorescens catalyzes the reaction of aromatic aldehydes with methoxy and dimethoxy acetaldehyde and furnishes (R)-2-hydroxy-3-methoxy-1-arylpropan-1-one and (R)-2-hydroxy-3,3-dimethoxy-1-arylpropan-1-one in high yields and enantiomeric excess via acyloin linkage. Aromatic aldehydes and benzoins are converted into enamine-carbanion-like intermediates prior to carboligation.

Factors affecting the electrochemical regeneration of NADH by (2,2'-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complexes: impact on their immobilization onto electrode surfaces.

Complexes of the (2,2'-bipyridyl) (pentamethylcyclopentadienyl)-rhodium family ([Cp*Rh(bpy)Cl](+), which is actually hydrolyzed in the form of [Cp*Rh(bpy)H(2)O](2+) in aqueous medium) are suitable solution-phase mediators likely to regenerate nicotinamide cofactors associated to dehydrogenases involved in many biocatalytic applications. Their practical application as bioelectrocatalysts, e.g., in fine chemicals synthesis or biosensors, remains however restricted to their durable immobilization in an active form onto solid electrode surfaces. This paper reports some new observations on the electrocatalytic properties of this mediator towards NAD(+) reduction, notably the critical effect of pH and cofactor-to-mediator concentration ratio, and investigates the behavior of a series of ([Cp*Rh(bpy)Cl](+)) derivatives bearing various substituents on the bipyridine ligand in view of their subsequent integration in electrochemical bioreactors. It will be shown that such compounds containing S- or N- moieties (i.e., often used as precursors to functionalize electrode surfaces) lead to inactivation of the electrocatalyst because their interaction with the Rh center prevents the formation of the active rhodium hydride complex. It was thus necessary to find another strategy of immobilization, and we found that adsorption of [Cp*Rh(bpy)Cl](+) by π-stacking on single-walled carbon nanotubes is an effective mean to reach this goal, leading to efficient and stable catalytic responses for NAD(+) reduction. Preliminary electroenzymatic experiments in the presence of d-sorbitol dehydrogenase further point out the interest of this approach for bioelectrocatalysis purposes and provide the proof-of-concept for this immobilization strategy.

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles.

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co(2+) modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.

Carboxylic acid derivatives of histone deacetylase inhibitors induce full length SMN2 transcripts: a promising target for spinal muscular atrophy therapeutics.

INTRODUCTION: Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder. It is caused by homozygous absence of the survival motor neuron 1 (SMN1) gene. SMN2, which modulates the severity of the disease, represents a major target for therapy. The aim of this study was to investigate whether SMN2 expression can be increased by caffeic acid, chlorogenic acid and curcumin, which are designed by modifications of the carboxylic acid class of histone deacetylase (HDAC) inhibitors. MATERIAL AND METHODS: Using quantitative real-time PCR, we analysed the levels of full-length SMN2 and Δ7SMN2 mRNA. We performed LDH cytotoxicity assay to analyse whether SMN2 activating concentrations of caffeic acid, chlorogenic acid and curcumin were cytotoxic to fibroblasts. RESULTS: We found that caffeic acid and curcumin were more efficient than chlorogenic acid and increased full-length SMN2 mRNA levels 1.5 and 1.7-fold, respectively. Δ7SMN2 mRNA levels were measured to investigate alternative splicing of exon 7. We also found that cytotoxicity was not observed at SMN2 activating concentrations. CONCLUSIONS: Our data suggest that carboxylic acid derivatives including phenolic structure and symmetry could be a good candidate for SMA treatment.

Chemoenzymatic conversion of linoleic acid into conjugated linoleic acid.

An efficient chemoenzymatic method for preparing conjugated linoleic acid (CLA) using free linoleic acid (LA) as a substrate is described. In the first step, LA was transformed into 10-hydroxy-cis-12-octadecenoic acid (HA) by the whole cells of Lactobacillus plantarum after 48 h of incubation. The preincubation of whole cells with 0.03% LA resulted in a better yield of HA (480 mg/g) compared to cells grown without LA. In a second fast microwave step, HA was converted to cis-9,trans-11-octadecadienoic acid in the presence of iodine as a catalyst over a silica gel surface. The advantage of this method in preparing cis-9,trans-11 CLA is simple via the whole cell bioconversion of LA into HA via L. plantarum followed by the fast microwave-assisted synthesis of cis-9,trans-11 CLA in higher yields.

Gold(I)/Zn(II) catalyzed tandem hydroamination/annulation reaction of 4-yne-nitriles.

The tandem hydroamination-annulation reaction of 4-pentyne-nitriles in the presence of amine nucleophiles and a cooperatively operating catalyst system, consisting of Ph(3)PAuCl and Zn(ClO(4))(2), provides an efficient route to 2-aminopyrroles. Two regioisomeric 2-aminopyrroles were formed in moderate to good yields.

Understanding the mode of action of ThDP in benzoylformate decarboxylase.

The mechanism of all elementary steps involved in the catalytic cycle of benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) to generate the acyloin linkage is investigated by extensive molecular dynamics simulations. Models involving different charge states of amino acids and/or mutants of critical residues were constructed to understand the involvement of the catalytically active residues and the reactivity differences between different substrates in this reaction. Our calculations confirm that H70, S26, and H281 are catalytically active amino acids. H281 functions as a base to accept H(donor) in the first nucleophilic attack and as an acid in the second, to donate the proton back to O(acceptor). S26 assists H281 in deprotonation of the donor aldehyde and protonation of the acceptor aldehyde. In both the first and second nucleophilic attacks, H70 interacts with O(aldehyde) and aligns it toward the nucleophilic center. H70 has been found to have an electrostatic effect on the approaching aldehyde whose absence would block the initiation of the reaction. The reactivity difference between benzaldehyde (BA) and acetaldehyde (AA) is mainly explained by the steric interactions of the acceptor aldehyde with the surrounding amino acids in the active center of the enzyme.

Histone deacetylase inhibition activity and molecular docking of (e )-resveratrol: its therapeutic potential in spinal muscular atrophy.

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )-resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration-dependent manner and half-maximum inhibition was observed at 650 microM. Molecular docking studies showed that (E )-resveratrol had more favorable free energy of binding (-9.09 kcal/mol) and inhibition constant values (0.219 microM) than known inhibitors. To evaluate the effect of (E )-resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )-resveratrol. The level of full-length SMN2 mRNA and protein showed 1.2- to 1.3-fold increase after treatment with 100 microM (E )-resveratrol in only one cell line. These results indicate that response to (E )-resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )-resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.

Addition of trifluoromethyltrimethylsilane to acyl phosphonates: synthesis of TMS-protected 1-alkyl-1-trifluoromethyl-1-hydroxyphosphonates and 1-aryldifluoroethenyl phosphates.

Addition reactions of nucleophilic CF3TMS to acyl phosphonates were investigated. Various acyl phosphonates reacted readily with CF3TMS in the presence of K2CO3 in DMF at rt to give 1-alkyl-2,2,2-trifluoro-1-trimethylsilyloxyethylphosphonate in 70-90% yields. When benzoyl phosphonates were used as starting material, after addition of CF3, the formed alcoholate undergoes phosphonate-phosphate rearrangement to form the acyl anion, followed by elimination of F- to give 1-aryldifluoroethenyl phosphates in 87-97% yields. As a representative example, vinylphosphate 6a was converted into 2,2-difluoro-1-phenylethanone 7 with 6 N HCl/EtOH/reflux or CAN/NaOH/MeOH/0 degrees C in 82-90% yields.

Origin of diastereoselectivity in the synthesis of chiral bicyclic lactams: pi-facial selective attack of singlet oxygen induced by hindered internal rotation.

Previously reported experimental results indicate that photooxygenation of homochiral N-(hydroxyalkyl)-2-methylpyrroles with singlet oxygen yields trans-rather than cis-bicyclic lactams as the major product. In this study, the origin of selectivity in this reaction has been investigated with computational methods. Relative stabilities of homochiral N-(hydroxyalkyl)-2-methylpyrrole conformers and their effect on pi-facial selectivity of 1O2 were extensively studied. Stepwise and concerted reaction mechanisms, starting from the endoperoxide intermediates, were proposed and modeled in vacuum using the UB3LYP method with the 6-31+G** basis set. Solvent calculations were carried out in CH2Cl2, by means of the integral equation formalism-polarizable continuum model (IEF-PCM) at the UB3LYP/6-31+G** level of theory. Free energies of activation leading to both diastereomers were analyzed in an effort to explain the stereoselectivity and product distribution. Steric interactions among the pyrrole substituents were shown to lead to a rotational barrier higher than 10 kcal/mol. Hence, hindered internal rotation is suggested to cause one pyrrole conformer to be substantially overpopulated. This in turn has a major effect on pi-facial selectivity of 1O2, thereby favoring one endoperoxide over the other and leading to the diastereoselective synthesis of trans-pyrrolooxazolones. The importance of hindered internal rotors, for an accurate calculation of the frequency factors of a chemical reaction, has already been mentioned in the literature many times; however, in this work hindered internal rotors also seem to dictate the diastereoselective outcome of the reaction.