Discovery of DSP-1053, a novel benzylpiperidine derivative with potent serotonin transporter inhibitory activity and partial 5-HT1A receptor agonistic activity.

We have previously shown that SMP-304, a serotonin uptake inhibitor with weak 5-HT1A partial agonistic activity, may act under high serotonin levels as a 5-HT1A antagonist that improves the onset of paroxetine in the rat swimming test. However, SMP-304 is mostly metabolized by CYP2D6, indicating limited efficacy among individuals and increased side effects. To reduce CYP2D6 metabolic contribution and enhance SERT/5-HT1A binding affinity, we carried out a series of substitutions at the bromine atom in the left part of the benzene ring of SMP-304 and replaced the right part of SMP-304 with a chroman-4-one. This optimization work led to the identification of the antidepressant candidate DSP-1053 as a potent SERT inhibitor with partial 5-HT1A receptor agonistic activity. DSP-1053 showed low CYP2D6 metabolic contribution and a robust increase in serotonin levels in the rat frontal cortex.

Regioselective trifluoromethylation of N-heteroaromatic compounds using trifluoromethyldifluoroborane activator.

Many important drugs, agrochemicals and their lead compounds contain trifluoromethyl group(s). Most processes currently used to access trifluoromethyl group-containing molecules are performed by substitution of the carboxy or trichloromethyl groups using hazardous fluorinating reagents under harsh reaction conditions. Cross-coupling reactions between organohalides or boronic acids/esters and trifluoromethylating reagents are also used. Direct C-H trifluoromethylation of organic molecules, however, is the ideal method of introducing trifluoromethyl group(s). Despite the recent advances in C-H trifluoromethylation of N-heteroaromatic compounds, regioselective C-H trifluoromethylation of six-membered heteroaromatic compounds has yet to be achieved. Herein we present a general and reliable method for the synthesis of trifluoromethyl group-containing N-heteroaromatics through highly regioselective addition of a trifluoromethyl nucleophile to pyridine, quinoline, isoquinoline and two or three heteroatom-containing N-heteroaromatic N-oxides activated by trifluoromethyldifluoroborane. The C-H trifluoromethylation proceeds under mild conditions in gram scale with high functional group tolerance. This method will be useful in both laboratory and industrial processes.

Synthesis of pyridine N-oxide-BF2CF3 complexes and their fluorescence properties.

Pyridine N-oxide-BF2CF3 and -BF2C2F5 complexes and their derivatives were synthesized. Most of the complexes show fluorescence both in solution and in the solid state. By expanding the π-conjugated skeleton, the color of the fluorescence could be changed dramatically. A fluorophore with a high solvent dependency could also be produced. Since such compounds can be synthesized on a gram scale in high yield, and are stable to oxygen, water, and heat, the complexes hold great potential as organic functional materials.

Degradation of triphenylborane-pyridine antifouling agent in water by copper ions.

Triphenylborane-pyridine (TPBP) is an antifouling compound used in Asian countries, including Japan, and its residue has not been detected in aquatic environments to date. There are limited data on its fate for environmental management. The purpose of this study was to evaluate whether TPBP is degraded by metal ions in aquatic environments. TPBP with metal ions in 20 mM sodium acetate buffer at pH 8.0 was placed at 25 degrees C in the dark for 24 h. The concentrations of TPBP and its degradation products, such as diphenylboronic acid, phenylboronic acid (MPB), phenol, benzene, biphenyl, and boron were determined. The presence of copper ions (50 mg/l), but not zinc or manganese ions, resulted in complete degradation of TPBP in 24 h. The TPBP degradation was much faster than the boron production in the initial reaction (0-1 h) with copper salts, depending on the copper salts tested. TPBP was degraded by copper ions (5 mg/l) in 24 h, producing phenol, MPB, biphenyl, and borate. Cu2+ as copper(II) chloride or copper(II) acetate led to complete degradation of TPBP, and thylenediaminetetraacetic acid disodium salt addition suppressed the TPBP degradation. Cu+ as copper(I) acetate also completely degraded TPBP, and bathocuproine addition suppressed the TPBP degradation. This suggests that copper ions existing in natural environments might degrade TPBP released from antifouling paint into water, and this could be one of the important mechanisms to dissipate TPBP residues in aquatic environments.

Effects of metal pyrithione antifoulants on freshwater macrophyte Lemna gibba G3 determined by image analysis.

Copper pyrithione (CuPT(2)) and zinc pyrithione (ZnPT(2)) are two popular antifouling agents that prevent biofouling. Research into the environmental effects of metal pyrithiones has mainly focused on aquatic animal species such as fish and crustaceans, and little attention has been paid to primary producers. There have been few reports on residues in environmental matrices because of the high photolabile characteristics of the agents. Residue analyses and ecological effects of the metabolites and metal pyrithiones are not yet fully understood. This study was undertaken to assess the effects of CuPT(2), ZnPT(2), and six metabolites (PT(2): 2,2'-dithio-bispyridine N-oxide, PS(2): 2,2'-dithio-bispyridine, PSA: pyridine-2-sulfonic acid, HPT: 2-mercaptopyridine N-oxide, HPS: 2-mercaptopyridine, and PO: pyridine N-oxide) on a freshwater macrophyte. A 7-day static bioassay using axenic duckweed Lemna gibba G3 was performed under laboratory conditions. Toxic effects of test compounds were assessed by biomass reduction and morphological changes were determined in image analysis. Concentrations of ZnPT(2) and CuPT(2) and those of PT(2) and HPT in the medium were determined by derivatizing 2,2'-dithio-bispyridine mono-N-oxide with pyridine disulfide/ethylene diamine tetra-acetic acid reagent that was equimolar with pyrithione. The toxic intensity of the compounds was calculated from the measured concentrations after 7-day exposure. ZnPT(2), CuPT(2), PT(2), and HPT inhibited the growth of L. gibba with EC(50) ranging from 77 to 140 µg/l as calculated from the total frond number as the conventional index, whereas the other four metabolites had less effect even at 10 mg/l. The presence of the former four toxic derivatives resulted in abnormally shaped and unhealthily colored fronds, whose size was about 20% of the control fronds. EC(50), calculated from the healthy frond area determined in image analysis, ranged from 10 to 53 µg/l. Thus, image analysis as part of a duckweed bioassay can detect the toxic effects of pyrithione derivatives with 3-10 times higher sensitivity than the traditional index.

Treatment of tetracycline antibiotics by laccase in the presence of 1-hydroxybenzotriazole.

Tetracycline antibiotics are widely used in human and veterinary medicine; however, residual amounts of these antibiotics in the environment are of concern since they could contribute to selection of resistant bacteria. In this study, tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) and oxytetracycline (OTC) were treated with laccase from the white rot fungus Trametes versicolor in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). High performance liquid chromatography demonstrated that DC and CTC were completely eliminated after 15 min, while TC and CTC were eliminated after 1 h. This system also resulted in a complete loss of inhibition of growth of Escherichia coli and Bacillus subtilis and the green alga Pseudokirchneriella subcapitata with decreasing tetracycline antibiotic concentration. These results suggest that the laccase-HBT system is effective in eliminating tetracycline antibiotics and removing their ecotoxicity.

Elimination of carbamazepine by repeated treatment with laccase in the presence of 1-hydroxybenzotriazole.

Carbamazepine (CBZP) is used as an antiepileptic drug and is highly persistent. In this study, CBZP was treated with laccase from white rot fungus Trametes versicolor in the presence of a redox mediator 1-hydroxybenzotriazole (HBT). A single treatment with laccase and HBT eliminated CBZP by about 22% after 24h, and repeated treatments with laccase and HBT, which were added to the reaction mixture every 8h, helped eliminate about 60% of CBZP after 48h. This suggests that repeated treatment is effective in eliminating CBZP. Mass spectrometric analyses demonstrated that two degradation products of CBZP, 10,11-dihydro-10,11-epoxycarbamazepine and 9(10H)-acridone, were formed via repeated treatment with laccase and HBT.

Elimination and detoxification of triclosan by manganese peroxidase from white rot fungus.

The antimicrobial and preservative agent triclosan (TCS) is an emerging and persistent pollutant with a ubiquitous presence in the aquatic environment. Thus, TCS was treated with manganese peroxidase (MnP), laccase and the laccase-mediator system with 1-hydroxybenzotriazole. MnP was most effective in eliminating TCS among the three enzymatic treatments, with TCS concentration being reduced by about 94% after 30 min following treatment with 0.5 nkat mL(-1) MnP and being almost completely eliminated after 60 min. Furthermore, MnP (0.5 nkat mL(-1)) caused the complete loss of bacterial growth inhibition by TCS after 30 min and reduced the algal growth inhibition of TCS by 75% and 90% after 30 and 60 min, respectively. These results strongly suggest that MnP is effective in removing the ecotoxicity of TCS.

Removal of diclofenac and mefenamic acid by the white rot fungus Phanerochaete sordida YK-624 and identification of their metabolites after fungal transformation.

The non-steroidal anti-inflammatory drugs diclofenac (DCF) and mefenamic acid (MFA) were treated with the white rot fungus Phanerochaete sordida YK-624. DCF completely disappeared and MFA decreased by about 90% after 6 days of treatment. It was also confirmed that the fungus almost completely removed the acute lethal toxicity of DCF and MFA towards the freshwater crustacean Thamnocephalus platyurus after 6 days of treatment. Mass spectrometric and (1)H nuclear magnetic resonance analyses demonstrated that two mono-hydroxylated DCFs (4'-hydroxydiclofenac and 5-hydroxydiclofenac) and one di-hydroxylated DCF (4',5-dihydroxydiclofenac) were formed via fungal transformation. The four metabolites of MFA were identified as 3'-hydroxymethylmefenamic acid (mono-hydroxylated MFA), 3'-hydroxymethyl-5-hydroxymefenamic acid (di-hydroxylated MFA), 3'-hydroxymethyl-6'-hydroxymefenamic acid (di-hydroxylated MFA) and 3'-carboxymefenamic acid. These results suggest that hydroxylation catalyzed by cytochrome P450 (CYP) in P. sordida YK-624 may be involved in the elimination and detoxification of DCF and MFA. This notion was further supported by the fact that smaller decreases in DCF and MFA were observed in cultures of P. sordida YK-624 incubated with 1-aminobenzotriazole, a known inhibitor of CYP.

Cloning and homologous expression of novel lignin peroxidase genes in the white-rot fungus Phanerochaete sordida YK-624.

Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.

Removal of estrogenic activity of iso-butylparaben and n-butylparaben by laccase in the presence of 1-hydroxybenzotriazole.

In the presence of a redox mediator, 1-hydroxybenzotriazole (HBT), iso-butylparaben (iso-BP) and n-butylparaben (n-BP) were treated with laccase from white rot fungus Trametes versicolor. HPLC analysis demonstrated that iso-BP and n-BP almost completely disappeared from the reaction mixture after 4 h of treatment with the laccase-HBT system. Using the yeast two-hybrid assay system, it was also confirmed that the laccase-HBT system substantially removed the estrogenic activity of iso-BP and n-BP after 4 h of treatment. Furthermore, there was a linear relationship between the removal of estrogenic activity of both parabens and the decrease in their concentrations. These results demonstrate that the laccase-HBT system is effective in eliminating iso-BP and n-BP, and removing the estrogenic activity of both parabens.

Efficient expression of laccase gene from white-rot fungus Schizophyllum commune in a transgenic tobacco plant.

Ligninolytic enzymes produced by white-rot fungi are effective degraders of recalcitrant aromatic environmental pollutants. However, gene sequences of these enzymes are rich in CpG dinucleotides, which are particularly unfavorable to efficient expression in plants. In order to develop a phytoremediation technique with a ligninolytic enzyme-producing transgenic plant, laccase cDNA (scL) from white-rot fungus Schizophyllum commune was used as a model ligninolytic enzyme, and we attempted to obtain the efficient expression of scL in a transgenic tobacco plant by decreasing the CpG-dinucleotide motif content. We constructed a mutagenized scL sequence, scL12, decreasing the CpG-dinucleotide motif content by 12%, and scL12 was introduced into the tobacco plant. Much higher laccase activity was detected in transgenic scL12 plants than in transgenic scL plants and wild-type plants. Using reverse transcriptase-PCR analysis, scL12 was translated in transgenic scL12 plants whereas mRNA of scL was not detected in the transgenic scL plants, and scL, which is the product of the scL12 gene, was produced in the transgenic scL12 plants using native-polyacrylamide gel electrophoresis analysis. Moreover, transgenic scL12 plants were able to remove trichlorophenol more effectively than transgenic scL plants and wild-type plants. These results suggest that decreasing CpG-dinucleotide motif content in fungal target genes is a useful method for efficient expression of these genes in transgenic plants.

Removal of estrogenic activity of 4-tert-octylphenol by ligninolytic enzymes from white rot fungi.

4-tert-Octylphenol (4-t-OP) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. 4-t-OP completely disappeared after 5 days of treatment and the activities of ligninolytic enzymes, laccase and manganese peroxidase (MnP), were detected during this period, thus suggesting that the disappearance of 4-t-OP is related to these extracellular enzymes. Therefore, 4-t-OP was treated with laccase and MnP prepared from white rot fungi cultures. HPLC analysis demonstrated that 4-t-OP completely disappeared in the reaction mixture after 1 h of treatment with either laccase or MnP. Using the yeast two-hybrid assay system, it was also confirmed that laccase and MnP substantially removed the estrogenic activity of 4-t-OP after 1 and 2 h of treatment, respectively. These results strongly demonstrate that ligninolytic enzymes are effective in removing the estrogenic activity of 4-t-OP.

Isolation and characterization of a novel bacterium, Sphingomonas bisphenolicum strain AO1, that degrades bisphenol A.

Bisphenol A (2,2-bis(4-hydroxyphenyl) propane, BPA), which is used as a synthetic resin material or a plasticizer, is a pollutant that possesses endocrine-disrupting activity. Bioremediation of BPA is used to decrease its polluting effects, and here we report a novel bacterial strain AO1, which is able to degrade BPA. This strain was isolated using enrichment cultivation from a soil sample from a vegetable-growing field; the sample was one of 500 soil samples collected across Japan. Strain AO1 degraded 100 mg/l BPA to an undetectable level within 6 h in MYPG medium (containing malt extract, yeast extract, peptone, and glucose) and within 48 h in minimum medium containing 1% glucose at 30 degrees C. Strain AO1 can utilize BPA as a sole source of carbon and as an energy source under aerobic conditions. The estrogenic activity of BPA in MYPG medium was ultimately reduced by strain AO1, although the activity initially increased. Taxonomical analysis showed that strain AO1 is closely related to Sphingomonas chlorophenolicum and S. herbicidovorans, neither of which have a capacity for BPA degradation. DNA-DNA hybridization showed that strain AO1 is a novel species of the Sphingomonas genus, and we designated AO1 as S. bisphenolicum.

Role of 1-hydroxybenzotriazole in oxidation by laccase from Trametes versicolor. Kinetic analysis of the laccase-1-hydroxybenzotriazole couple.

In the current studies, we used Lineweaver-Burke analysis to examine the role of 1-hydroxybenzotriazole (HBT) in the oxidation of various compounds by laccase from Trametes versicolor. At low concentrations, HBT was a competitive inhibitor of the oxidation, but at high concentrations, it was a noncompetitive inhibitor. Analysis of the oxidation of ferrocytochrome c by the laccase-HBT couple showed that increasing the concentration of ferrocytochrome c did not affect the V(max) but reduced the apparent K(m). In addition, in the manganese peroxidase-Mn(II) reaction, which is a typical oxidation system by mediator, the apparent K(m) and V(max) increased as the concentration of the substrate 2,6-dimethoxyphenol was increased. These results indicate that HBT is involved in the binding of laccase and substrates that laccase cannot oxidize alone.

The cationic cell-wall-peroxidase having oxidation ability for polymeric substrate participates in the late stage of lignification of Populus alba L.

Previously we reported that purified Cell Wall Peroxidase-Cationic (CWPO-C) from poplar callus (Populus alba L.) oxidizes sinapyl alcohol and polymeric substrate unlike other plant peroxidases and proposed that this isoenzyme is a conceivable lignification specific peroxidase. In this study, we cloned full-length cDNA of CWPO-C and investigated the transcription of CWPO-C gene in various organs and the localization of CWPO-C protein in the differentiating xylem of poplar stem.Real-time PCR analyses indicated that CWPO-C gene is constitutively expressed in the developing xylem, leaf, and shoot but not affected by many stress treatments. Immunohistochemical analysis showed that CWPO-C locates in the middle lamellae, cell corners, and secondary cell walls of the fiber cells during the lignification. The intensity of the CWPO-C labeling increased gradually from the cell wall thickening stage to mature stage of fiber cells, which is very consistent with the increase of lignin content in the developing xylem. These results strongly support that CWPO-C is responsible for the lignification of the secondary xylem. Interestingly, immuno-labeling of CWPO-C was also observed inside of the ray parenchyma cells instead no signals were detected within the developing fiber cells. This suggests that CWPO-C is biosynthesized in the parenchyma cells and provided to the middle lamellae, the cell corners, and the cell walls to achieve lignin polymerization.

Removal of estrogenic activity of natural steroidal hormone estrone by ligninolytic enzymes from white rot fungi.

Natural steroidal hormone estrone (E1) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. E1 decreased by 98% after 5 d of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, which suggested that the disappearance of E1 is related to ligninolytic enzymes produced extracellularly by white rot fungus. Therefore, E1 was treated with MnP and laccase prepared from the culture of white rot fungi. HPLC analysis demonstrated that E1 disappeared completely in the reaction mixture after 1 h of treatment with either MnP or laccase. Using the yeast two-hybrid assay system, it was also confirmed that both enzymatic treatments completely removed the estrogenic activity of E1 after 2 h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of E1.

Characteristics of novel lignin peroxidases produced by white-rot fungus Phanerochaete sordida YK-624.

We characterized a lignin peroxidase (YK-LiP2) isolated from shaking culture inoculated with the white-rot fungus Phanerochaete sordida YK-624. The YK-LiP2 enzyme was identified and purified to homogeneity by anion-exchange chromatography and gel permeation chromatography. The molecular weight of YK-LiP2 was approximately 45 kDa, and its absorption spectrum was almost the same as that of the LiP (Pc-LiP) from P. chrysosporium. Steady-state kinetics of veratryl alcohol (VA) oxidation by YK-LiP2 revealed an ordered bi-bi ping-pong mechanism, although the Pc-LiP oxidation of ferrocytochrome c obeys peroxidase ping-pong kinetics rather than ordered bi-bi ping-pong kinetics. Degradation of dimeric lignin model compounds by YK-LiP2 was more effective than that by Pc-LiP. Moreover, YK-LiP2 and YK-LiP1, which was previously isolated from static culture inoculated with P. sordida YK-624, oxidized VA under a higher concentration of hydrogen peroxide (>2.5 mM) although Pc-LiP could not oxidize VA in the presence of 2.5 mM hydrogen peroxide.

Removal of estrogenic activity of endocrine-disrupting genistein by ligninolytic enzymes from white rot fungi.

Endocrine-disrupting genistein was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. Genistein decreased by 93% after 4 days of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, thus suggesting that the disappearance of genistein is related to ligninolytic enzymes produced extracellularly by white rot fungi. Therefore, genistein was treated with MnP, laccase, and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as a mediator. HPLC analysis demonstrated that genistein disappeared almost completely in the reaction mixture after 4 h of treatment with either MnP, laccase, or the laccase-HBT system. Using the yeast two-hybrid assay system, it was also confirmed that three enzymatic treatments completely removed the estrogenic activity of genistein after 4h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of genistein.

Three-component coupling based on the "cation pool" method.

Sequential one-pot three-component coupling reactions have been developed based on the "cation pool" method. An N-acyliminium ion generated by the "cation pool" method adds to an electron-rich carbon-carbon double bond, such as enamine derivatives and vinyl sulfides, to form the second "cation pool". The addition of nucleophiles such as allylsilanes, enol silyl ethers, Grignard reagents, and organoaluminum compounds led to the formation of the corresponding three-component coupling products.