Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus.

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains.

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Biodegradation of pentachlorophenol in natural soil by inoculatedRhodococcus chlorophenolicus

Rhodococcus chlorophenolicus PCP-1, a mineralizer of polychlorinated phenols, was inoculated into natural sandy loam and peaty soils with pentachlorophenol (PCP) at concentrations usually found at lightly and heavily polluted industrial sites (30 to 600 mg PCP/kg). A single inoculum of 10⁵ to 10⁸ cells per g of peat soil and as little as 500 cells/g sandy soil initiated mineralization of¹⁴C-PCP. The mineralization rates of PCP were 130 to 250 mg mineralized per kg soil in 4 months in the heavily (600 mg/kg) polluted soils and 13 to 18 mg/kg in the lightly (30 mg/kg) polluted soils. There were no detectable PCP mineralizing organisms in the soils prior to inoculation, and also there was no significant adaptation of the indigenous microbial population to degrade PCP during 4 months observation in the uninoculated soils. The inoculum-induced mineralization continued for longer than 4 months after a single inoculation. Uninoculated, lightly polluted soils (30 mg PCP/kg) also showed loss of PCP, but some of this reappeared as pentachloroanisol and other organic chlorine compounds (EOX). Such products did not accumulate in theR. chlorophenolicus-inoculated soils, where instead EOX was mineralized 90 to 98%.R. chlorophenolicus mineralized PCP unhindered by the substrate competition offered by the PCP-methylating bacteria indigenously occurring in the soils or by simultaneously inoculated O-methylatingR. rhodochrous.     

Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus

Summary An actinomycete, Rhodococcus chlorophenolicus, isolated from a pentachlorophenol-degrading mixed bacterial culture is a polychlorophenol degrader. It was shown to oxidize pentachlorophenol into carbon dioxide and to metabolize also 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6-, and 2,4,5-trichlorophenol, 2,5-, and 2,6-dichlorophenol and tetrachloro-p-hydroquinone in an inducible manner. Pentachlorophenol set on the synthesis of enzymes required for the metabolism of all these chlorophenols and of tetrachloro-p-hydroquinone. 2,4,5-, and 2,4,6-trichlorophenol and 2,5-, and 2,6-dichlorophenol were degraded by R. chlorophenolicus cells only if these had previous contact to pentachlorophenol. Other chlorophenols mentioned were able to set on the synthesis of enzymes for their own degradation. 2,3,4,5-, and 2,3,4,6-tetrachlorophenol, and 2,3,5-, 2,4,5-, and 3,4,5-trichlorophenol were more toxic to R. chlorophenolicus than the other chlorophenols, but nevertheless 2,3,4,5-, and 2,3,4,6-tetrachlorophenol and 2,3,5-trichlorophenol were readily degraded by the bacteria.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - TeCH tetrachloro-p-hydroquinone An example of numeration: 2345-TeCP, 2,3,4,5-tetrachlorophenol     

The survival of the pentachlorophenol-degrading Rhodococcus chlorophenolicus PCP-1 and Flavobacterium sp. in natural soil

The survival of two different pentachlorophenol (PCP)-degrading bacteria were studied in natural soil. The PCP-degraders Rhodococcus chlorophenolicus and Flavobacterium sp., both able to mineralize PCP into CO₂ and chloride in axenic culture, were tested for the capacity to survive and degrade PCP in natural soil. These bacteria were immobilized on polyurethane (PUR) foam and introduced into natural peaty soil to give about 10⁹ cells g^-1 of soil (dry weight). R. chlorophenolicus induced PCP-degrading activity in soil remained detectable for 200 days whether or not a carbon source was added (distillery waste or wood chips). Electron microscopic investigation performed almost a year after inoculation, revealed the presence of R. chlorophenolicus-like cells in the PUR foam particles. PCP-degrading activity of Flavobacterium sp. declined within 60 days of burial in the soil without enhancing the PCP removal. R. chlorophenolicus degraded PCP in soil at a mean rate of 3.7 mg of PCP day^-1 kg^-1 of soil, which corresponds to ca. 5×10^-3 pg of PCP degraded per inoculated R. chlorophenolicus cell day^-1. The solvent extractable organic chlorine contents of the soil decreased stoichiometrically (>95%) with that of PCP indicating that PCP was essentially mineralized.Abbreviations ATCC American type culture collection - DSM Deutsche Sammlung für Mikroorganismen - DW distillery waste - EM electron microscopy - EOX extractable organic halogen - GC/ECD gas chromatograph/electron capture detector - GC/MS gas chromatograph/mass spectrometer - PCP pentachlorophenol - WC wood chips - d.wt. dry weight - w.wt. wet weight - d.s. dry soil - d.H₂O distilled water - PCA polychlorinated aromatics     

Aerobic Mineralization of 2,6-Dichlorophenol by Ralstonia sp. Strain RK1

A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP) as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 μM (50 mg · liter⁻¹), 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 μM in continuous reactor culture supplied with H₂O₂ as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 μM) with hydraulic residence times of 8 to 30 min.     

Effect of Chlorine Substitution on the Biodegradability of Polychlorinated Biphenyls

Thirty-one isomers of polychlorinated biphenyl (PCB) were examined for biodegradability by two species of Alcaligenes and Acinetobacter. The following relationships between chlorine substitution and biodegradability of PCBs were observed. (i) Degradation decreased as chlorine substitution increased. PCB isomers containing more than four chlorines were less susceptible to degradation. (ii) PCBs containing two chlorines on either the ortho position of a single ring (i.e., 2,6-) or on both rings (i.e., 2,2′-) showed very poor degradability. (iii) PCBs containing all chlorine atoms on only a single ring were generally degraded faster than when the same number of chlorines were substituted on both rings. (iv) Preferential ring fission of the molecules occurred with nonchlorinated or lesser chlorinated rings. (v) The formation and accumulation of a yellow intermediate was always observed in 4′-chloro-substituted PCBs. (vi) Significant differences between the two organisms with respect to degradability were not observed except for 2,4,6-trichlorobiphenyl.     

Novel 4-Chlorophenol Degradation Gene Cluster and Degradation Route via Hydroxyquinol in Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (cph genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes, cphA-I and cphA-II, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the cphA-I gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the cph gene cluster to functional catabolism of 4-chlorophenol in A. chlorophenolicus A6. The presence of a resolvase pseudogene in the cph gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.     

Brominated Biphenyls Prime Extensive Microbial Reductive Dehalogenation of Aroclor 1260 in Housatonic River Sediment

The upper Housatonic River and Woods Pond (Lenox, Mass.), a shallow impoundment on the river, are contaminated with polychlorinated biphenyls (PCBs), the residue of partially dechlorinated Aroclor 1260. Certain PCB congeners have the ability to activate or “prime” anaerobic microorganisms in Woods Pond sediment to reductively dehalogenate the Aroclor 1260 residue. We proposed that brominated biphenyls might have the same effect and tested the priming activities of 14 mono-, di-, and tribrominated biphenyls (350 μM) in anaerobic microcosms of sediment from Woods Pond. All of the brominated biphenyls were completely dehalogenated to biphenyl, and 13 of them primed PCB dechlorination. Measured in terms of chlorine removal and decrease in the proportion of hexa- through nonachlorobiphenyls, the microbial PCB dechlorination primed by several brominated biphenyls was nearly twice as effective as that primed by chlorinated biphenyls. Congeners containing a meta bromine primed Dechlorination Process N (flanked meta dechlorination), and congeners containing an unflanked para bromine primed Dechlorination Process P (flanked para dechlorination). Two ortho-substituted congeners, 2-bromobiphenyl and 2,6-dibromobiphenyl (2-BB and 26-BB), also primed Process N dechlorination. The most effective primers were 26-BB, 245-BB, 25-3-BB, and 25-4-BB. The microbial dechlorination primed by 26-BB converted ~75% of the hexa- through nonachlorobiphenyls to tri- and tetrachlorobiphenyls in 100 days and removed ~75% of the PCBs that are most persistent in humans. These results represent a major step toward identifying an effective method for accelerating PCB dechlorination in situ. The challenge now is to identify naturally occurring compounds that are safe and effective primers.     

Microbial Anaerobic Demethylation and Dechlorination of Chlorinated Hydroquinone Metabolites Synthesized by Basidiomycete Fungi

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.     

Modified 2,4-diaryl-5H-indeno[1,2-b]pyridines with hydroxyl and chlorine moiety: Synthesis,anticancer activity,and structure–activity relationship study

As a part of ongoing studies in developing novel anticancer agents, a series of modified 2,4-diaryl-5H-indeno[1,2-b]pyridines were designed, and synthesized by introducing hydroxyl and chlorine moieties. They were evaluated for topoisomerase inhibitory activity and cytotoxicity against HCT15, T47D, and HeLa cancer cell lines. This modification allowed us to demonstrate structure–activity relationship (SAR) study with respect to the non-substituted 2,4-diaryl-5H-indeno[1,2-b]pyridines. Compounds (2, 3, 4, 5, 8, and 9) with meta or para hydroxyl group on 2 or 4-phenyl ring have enhanced topo I and II inhibitory activity and cytotoxicity. However, additional substitution of chlorine group on furyl or thienyl ring (11, 12, 14, 16–18) generally reduced topo I and II inhibitory activity but improved cytotoxicity. The observation of cytotoxic properties and SAR study according to the position of hydroxyl and chlorine group will provide valuable insight for further study of development of novel anticancer agents with related scaffolds.     

Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone

Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V_max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.     

Spectrum of the Reductive Dehalogenation Activity of Desulfitobacterium frappieri PCP-1

Desulfitobacterium frappieri PCP-1 was induced for ortho- and para-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho, meta, and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.     

Bacterial Methylation of Chlorinated Phenols and Guaiacols: Formation of Veratroles from Guaiacols and High-Molecular-Weight Chlorinated Lignin

Two strains of bacteria, provisionally assigned to the genus Arthrobacter, were shown to metabolize mono-, di-, tri-, and tetrachloroguaiacols and pentachlorophenol to the corresponding O-methyl compounds. Hydroxylated intermediates were formed only transiently, except for the synthesis by one strain of 3,4,5-trichlorosyringol from 3,4,5-trichloroguaiacol. Two isomeric trichloroveratroles and tetrachloroveratrole were formed by three of the strains from a high-molecular-weight chlorinated lignin isolated from kraft pulp mill bleach plant. The concentrations of methylated metabolites varied widely and did not appear to be correlated with degradation. The possible environmental consequences resulting from synthesis of these highly lipophilic substances are discussed briefly.     

Metabolism of chloroguaiacols by Rhodococcus chlorophenolicus

Summary A polychlorophenol degrader, Rhodococcus chlorophenolicus, was shown to metabolize five different chlorinated guaiacols, namely tetrachloroguaiacol, 3,4,6-trichloroguaiacol, 3,5,6-trichloroguaiacol, 3,5-dichloroguaiacol and 3,6-dichloroguaiacol. Seven different intermediate metabolites, each with three hydroxyl or methoxyl groups, were identified. Four of these metabolites were also dehalogenation products, three carrying one chlorine atom less than the parent compound, and one metabolite from tetrachloroguaiacol where two chlorine atoms had been removed. Tetrachloroguaiacol was shown to undergo reductive dehalogenation. Demethylation of guaiacol to catechol was observed with the dichloroguaiacols, but not with polychloroguaiacols.Abbreviations DCG dichloroguaiacol - TCG trichloroguaiacol - TeCG tetrachloroguaiacol - DCC dichlorocatechol - TCC trichlorocatechol - TeCC tetrachlorocatechol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol. An example of numeration - 346-TCG 3,4,6-trichloroguaiacol - GLC gas liquid chromatography     

Greenhouse evaluation of chemicals for control of powdery mildews

2,6-Dinitro-4-s-alkylphenols were found to protect apple foliage against powdery mildew more effectively than isomeric 2,4-dinitro-6-s-alkylphenols; regression lines for four para-alkyl compounds were of similar slope but were much steeper than those for two ortho-alkyl compounds (including dinocap phenol). The ED₅₀ and ED₉₅ values of the most active compound studied, 2,6-dinitro-4-(I-ethylhexyl)phenol, were in the ratios to those of dinocap phenol of 1:13 and 1:125, respectively, and the protective action of this compound was greater than the curative, especially at the higher ED values. Protection of barley seedlings against powdery mildew was also greater by I-ethyl- or I-propyl-alkyl compounds than by I-methylalkyl or n-alkyl isomers. For phytotoxic and acaricidal actions, the ortho-alkyl isomers are more effective than para-alkyl. Nevertheless, the acaricidal activity of dinocap phenol is exceeded by that of the isomeric 2,4-dinitro-6-(I-ethylhexyl) phenol. The control of these powdery mildew diseases given by commercial products, supposed to be based on dinocap, cannot be accounted for by the activity shown for dinocap phenol. It can, however, be accounted for by the activities of 2,6-dinitro-4-(I-ethylhexyl)- and -(I-propylpentyl)-phenols, now known to be present in commercial products in larger amounts than dinocap phenol itself. The phytotoxic and acaricidal actions of such products, however, are mainly due to the ortho-octyldinitrophenols present; in view of the small proportion that is dinocap phenol, the acaricidal activity is likely to be due almost entirely to the other ortho-octyl isomers. It is suggested that the common name dinocap be retained for the mixture of dinitrooctylphenols now known to be present in commercial products, and that the two main groups be differentiated as 2,4-dinocap and 2,6-dinocap, respectively. The advantages for powdery mildew control of a product based on 2,6-dinocap are discussed.     

Degradation of 2,4,6-Trichlorophenol by Phanerochaete chrysosporium: Involvement of Reductive Dechlorination

Under secondary metabolic conditions, the lignin-degrading basidiomycete Phanerochaete chrysosporium mineralizes 2,4,6-trichlorophenol. The pathway for the degradation of 2,4,6-trichlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway is initiated by a LiP- or MnP-catalyzed oxidative dechlorination reaction to produce 2,6-dichloro-1,4-benzoquinone. The quinone is reduced to 2,6-dichloro-1,4-dihydroxybenzene, which is reductively dechlorinated to yield 2-chloro-1,4-dihydroxybenzene. The latter is degraded further by one of two parallel pathways: it either undergoes further reductive dechlorination to yield 1,4-hydroquinone, which is ortho-hydroxylated to produce 1,2,4-trihydroxybenzene, or is hydroxylated to yield 5-chloro-1,2,4-trihydroxybenzene, which is reductively dechlorinated to produce the common key metabolite 1,2,4-trihydroxybenzene. Presumably, the latter is ring cleaved with subsequent degradation to CO₂. In this pathway, the chlorine at C-4 is oxidatively dechlorinated, whereas the other chlorines are removed by a reductive process in which chlorine is replaced by hydrogen. Apparently, all three chlorine atoms are removed prior to ring cleavage. To our knowledge, this is the first reported example of aromatic reductive dechlorination by a eukaryote.     

Selective benzylation of some D-galactopyranosides. Unusual relative reactivity of the hydroxyl group at C-4

Partial benzylation of methyl 2,3-di-O-benzyl-α-D-galactopyranoside gave methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside as the major product, whereas the isomeric 2,6-di-O-benzyl ether gave a mixture of products in which the ratio of methyl 2,4,6- to methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside was ≈4:1. The proportion of unreacted starting-material was low in both cases, whereas after a similar reaction of methyl 2,6-di-O-benzyl-β-D-galactopyranoside more than 50% of the dibenzyl ether was recovered unchanged. In this case also, considerably higher reactivity was exhibited by the hydroxyl group at C-4 than that at C-3. Acid hydrolysis of the methyl glycosides of the tribenzyl ethers afforded crystalline 2,4,6-tri-O-benzyl-α-D-galactose and syrupy 2,3,6-tri-O-benzyl-D-galactose. Structures of intermediates were established by acetylation, examination of their n.m.r. spectra, and conversion into the known 3-O and 4-O-methyl-D-galactose.