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Anaerobic Microbial Transformation of Halogenated Aromatics and Fate Prediction Using Electron Density Modelling Myriel Cooper, Anke Wagner, Dominik Wondrousch, Frank Sonntag, Andrei Sonnabend, Martin Brehm, Gerrit Schüürmann, and Lorenz Adrian Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00303 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
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Anaerobic Microbial Transformation of Halogenated Aromatics
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and Fate Prediction Using Electron Density Modelling
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Myriel Cooper,† Anke Wagner,‡,ǂ Dominik Wondrousch,§,ǁ Frank Sonntag,‡
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Andrei Sonnabend,‡ Martin Brehm,§ Gerrit Schüürmann,§,ǁ and Lorenz Adrian†,‡,*
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†
Helmholtz-Zentrum für Umweltforschung – UFZ, Isotope Biogeochemistry, Permoserstr. 15,
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04318 Leipzig, ‡Technische Universität Berlin, Applied Biochemistry, Gustav-Meyer-Allee 25,
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13355 Berlin, §Helmholtz-Zentrum für Umweltforschung – UFZ, Ecological Chemistry,
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Permoserstr. 15, 04318 Leipzig and ǁTechnische Universität Bergakademie Freiberg, Institute for
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Organic Chemistry, Leipziger Strasse 29, 09596 Freiberg, Germany
10 11
Keywords: microbiology, biodegradation, computational modeling, enzyme mechanism,
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chemical biology, Dehalococcoides, halogenated aromatics, reductive dehalogenation,
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organohalide respiration, B12
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*Corresponding author: Helmholtz Centre for Environmental Research – UFZ, Isotope
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Biogeochemistry, Permoserstraße 15, 04318 Leipzig, Germany, Tel.: +49 - 341 235 1435,
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e-Mail:
[email protected] 17
Running title: Fate of halogenated aromatics in anaerobic environments
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ABSTRACT
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Halogenated homo- and heterocyclic aromatics including disinfectants, pesticides and
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pharmaceuticals raise concern as persistent and toxic contaminants with often unknown fate.
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Remediation strategies and natural attenuation in anaerobic environments often build on
22
microbial reductive dehalogenation. Here we describe the transformation of halogenated anilines,
23
benzonitriles, phenols, methoxylated or hydroxylated benzoic acids, pyridines, thiophenes, furoic
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acids and benzenes by Dehalococcoides mccartyi strain CBDB1 and environmental fate
25
modelling of the dehalogenation pathways. The compounds were chosen based on structural
26
considerations to investigate the influence of functional groups present in a multitude of
27
commercially used halogenated aromatics. Experimentally obtained growth yields were 0.1-5 x
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1014 cells mol-1 of halogen released (corresponding to 0.3 to 15.3 g protein mol-1 halogen), and
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specific enzyme activities ranged from 4.5 to 87.4 nkat mg-1 protein. Chlorinated electron-poor
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pyridines were not dechlorinated in contrast to electron-rich thiophenes. Three different partial
31
charge models demonstrated that the regioselective removal of halogens is governed by the least
32
negative partial charge of the halogen. Microbial reaction pathways combined with
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computational chemistry and pertinent literature findings on CoI chemistry suggest that halide
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expulsion during reductive dehalogenation is initiated through single electron transfer from
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B12CoI to the apical halogen site.
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INTRODUCTION
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Halogenated aromatic hydrocarbons are widely used e.g. as solvents, biocides, flame retardants
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or additives to polymers. Commercialized halogenated aromatics often contain non-halogen
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substituents or heteroatoms in the cycle determining the physicochemical and biological
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properties in aerobic and anaerobic environments. Halogenated heterocycles can be extremely
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persistent and toxic as seen with the polyhalogenated dibenzodioxins and dibenzofuranes.
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A number of strictly anaerobic bacteria have been isolated using organohalogen compounds as
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electron acceptor in an anaerobic respiration including members of the genus Dehalococcoides.1-
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3
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Dehalococcoides mccartyi strain CBDB1 transforms a wide variety of halogenated aromatic
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compounds including benzenes, phenols, biphenyls and dioxins1,4-6 and uses the electron transfer
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for energy conservation.1
Whereas some Dehalococcoides strains are specialized on chlorinated ethenes,2,3
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Reductive dehalogenation of halogenated aromatic compounds by strain CBDB1 is not random
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but follows distinct rules, which allow to some extent the prediction of dehalogenation pathways.
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Halogen substituents on aromatic compounds have previously been classified as doubly flanked,
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singly flanked or isolated when they have two, one or no neighboring halogen substituent,
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respectively.7 Strain CBDB1 reductively dehalogenated doubly flanked substituents of
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chlorinated benzenes and polychlorinated biphenyls (PCBs) and with a lower rate also singly
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flanked substituents, whereas isolated chlorine substituents were not removed.4,8 It was also
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shown that strain CBDB1 removes doubly, singly and unflanked bromine substituents from
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bromobenzenes.9
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The hydroxyl group in chlorophenols enhanced dechlorination of a neighboring chlorine
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substituent similar to a chlorine substituent.5 Also halogen substituents singly flanked by one 3
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hydroxyl group were dechlorinated as shown for 2,4,5-trichlorophenol which was dechlorinated
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to 4,5-dichlorophenol. However, the effect of a hydroxyl-group was weaker than the effect of a
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chlorine substituent when it was in meta- or para-position.
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An enhancing effect on dechlorination was observed for the oxygen atom in the heteroaromatic
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ring of dioxins. 1,2,3-tri- and 1,2,3,4-tetrachlorodibenzo-p-dioxin were both dehalogenated to
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2,3-dichlorodibenzo-p-dioxin.6,10 Similarly 1,2,3,7,8-pentachlorodibenzo-p-dioxin was converted
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preferentially to the most toxic congener 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reaction that will
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also occur in nature from highly chlorinated dioxins. The peri-substituent in position 1 was
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preferred over the doubly flanked substituent in position 2.
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In a previous study, chemical properties of chlorobenzenes, dioxins and chlorophenols were
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modelled using density functional theory calculations at the B3LYP/6-31G(d) level.11 Mulliken
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partial charges were identified as a tool to describe the dehalogenation pathways of these three
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classes of chlorinated persistent organic pollutants catalyzed by strain CBDB1. Lu and
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colleagues correlated more positive partial charges of chlorine substituents with dehalogenation
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pathways of strain CBDB1 previously described in the literature. However, the Mulliken model
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does not account for differences in electronegativity of atoms within one molecule.12,13 In
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addition, due to the restricted number of available experimental data this model excludes most of
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the commercially used halogenated aromatics and molecules with two or more different halogen
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substituents.
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Reductive dehalogenases are the key catalysts for reductive dehalogenation.
In
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Dehalococcoides strains reductive dehalogenases are not specific for only one substrate or even
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one mechanism as has been shown e.g. for the TceA enzyme of strain 195,14 the BvcA enzyme
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of strain BAV115 and the VcrA enzyme of strain VS16. In strain CBDB1 very broad enzyme 4
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specificity has been shown towards many different halogenated aromatics by the CbrA
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enzyme.9,17,18 Reductive dehalogenases are mostly encoded by operons with two genes: the
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catalytic subunit (rdhA) and a small membrane protein proposed to function as a membrane
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anchor (rdhB).19 Generally, the catalytic subunits contain two binding motifs for iron-sulfur
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clusters and in some cases binding motifs for corrinoid cofactors.20 Although corrinoid binding
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motifs are rare in rdhA genes of Dehalococcoides strains light-reversible inhibition by alkyl-
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iodids indicated the involvement of a corrinoid cofactor in most dehalogenases studied up to
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now.18,21 All yet-purified reductive dehalogenases contained a corrinoid cofactor, with the
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exception of the 3-chlorobenzoate reductive dehalogenase of Desulfomonile tiedjei.22 On the
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basis of the fact that superreduced corrinoids are strong nucleophiles, several reaction
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mechanisms have been proposed including nucleophilic substitution19 and a mechanism with
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radical intermediates.23-25 Both mechanisms focus on the addition of electrons to the partially
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positively charged carbon atom from which the halogen substituent is removed. Very recently,
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the 3D-structures of a tetrachloroethene reductive dehalogenase from Sulfurospirillum
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multivorans26 and a soluble, oxygen-tolerant reductive dehalogenase from Nitratireductor
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pacificus pht-3B27 specialized on ortho–halogenated phenolic substrates were resolved providing
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a structural basis to understand organohalide respiration. For the oxygen-tolerant reductive
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dehalogenase a cobalt-halogen interaction during catalysis was suggested.27
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In this study, D. mccartyi strain CBDB1 was cultivated with a broad range of electron
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acceptors containing different heteroatoms (-O; -S; -N), halogen substituents (-Br; -Cl; -F) or
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non-halogen substituents (-NH2; -CN; -OH; -OCH3) to study their influence on reductive
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dehalogenation. Whole-cell activity assays were conducted to determine specific activities. Three
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partial charge models were analysed for their ability to predict dehalogenation pathways. Strain 5
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CBDB1 dehalogenates halogenated anilines, benzonitriles, pyridines, thiophenes, benzoic and
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furoic acids to a different extent. Functional groups enhance or inhibit reductive dehalogenation.
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Comparative analysis of the results together with corresponding data from previous
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investigations on reductive dehalogenation1,4-6,8 show that halogen net atomic charges, calculated
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by quantum chemistry, are good indicators for the regioselectivity of the observed
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dehalogenation pathways. Associated mechanistic reasoning strongly suggests a primary attack
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of B12CoI at the rear (apical) electron-poor site of the aromatically bound halogen followed by an
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inner-sphere electron-transfer reaction, which contrasts with previous mechanistic reaction
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models and suggests that the recently proposed halogen-cobalt interaction in an oxygen-tolerant
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cytoplasmic dehalogenase27 is also formed in membrane-bound respiratory reductive
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dehalogenases of strictly anaerobic bacteria. The results promote our ability to predict the
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regioselective dehalogenation of a wide range of halogenated aromatics which is important for
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the evaluation of environmental persistence and toxicity.
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EXPERIMENTAL SECTION
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Chemicals. Chlorinated and brominated aromatic compounds were purchased from Sigma-
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Aldrich (Seelze, Germany) at a purity of 99%. Titanium(III) citrate was prepared as previously
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described.5
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Cultivation. Strain CBDB1 was grown in titanium(III) citrate-reduced, carbonate-buffered,
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vitamin amended synthetic medium with hydrogen as electron donor and 5 mM acetate as carbon
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source, as previously described.1 After inoculation, the headspace was pressurized with 20% CO2
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/ 80% N2 (1.5 bar), and hydrogen was added to a pressure of 1.8 bar. Cultivation was performed
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in the dark at 30 °C without shaking. The electron acceptors were added from either 1 M stock 6
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solutions in acetone at a final concentration of 50 or 100 µM or as crystals (Supporting
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Information, Table S1). All cultures were initially set up from cultures grown on 30 µM 1,2,3-
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trichlorobenzene. A starting cell density of about 1 x 106 cell ml-1 was chosen. Cell numbers
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were quantified after staining with SYBR-green by direct cell counting on agarose-coated slides
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as previously described.5,28 All cultivations were done in duplicates or triplicates. Additionally,
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chemical controls without inoculum, negative growth controls without electron acceptor and
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positive controls with 30 µM 1,2,3-trichlorobenzene were set-up. Results presented are from
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cultures of the first or second transfer with the respective electron acceptor. First transfers are
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shown when one of the electron acceptors in a compound class did not support growth so that no
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second transfer was available.
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Analytical Techniques. 1,2,3-trichlorobenzene, chlorinated anilines, benzonitriles, thiophenes
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and pyridines were extracted from 0.5 ml samples with 1 ml of hexane and analyzed using a
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Shimadzu gas chromatograph (GC) 14A equipped with flame ionization detector on a
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Permabond-FFAP capillary column (25 m, 0.25 mm inner diameter, 0.25 µm film thickness,
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Macherey & Nagel, Düren, Germany). Nitrogen (99.999% purity) served as carrier and make-up
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gas. The following temperature program was used: initial hold at 55 °C for 1 min; increase at a
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rate of 6 °C min-1 to 225 °C, with a final hold for 2 min. Injector and detector temperatures were
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250 and 300 °C, respectively.
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Bromophenols from 1 ml culture volume were derivatized with 120 mM sodium bicarbonate
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and 50 mM acetic anhydride for 10 min on a rotary shaker set to 350 rpm at room temperature
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before extracting the sample with 1 ml hexane. Samples were analyzed using a Hewlett Packard
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GC 6890 equipped with flame ionization detector on a HP-5 capillary column (30 m, 0.25 mm
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inner diameter, 0.25 µm film thickness, Agilent Technologies, Böblingen, Germany). The 7
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temperature program started with an initial hold for 1 min at 55 °C and continued by an increase
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of 10 °C min-1 to 150 °C, then 10 °C min-1 to 230 °C, and 30 °C min-1 to 260 °C with a final hold
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of 2 min. Injector and detector temperatures were 250 °C.
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Thiophene was analyzed from the headspace of the culture. Two hundred µl of the headspace
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were removed at each time point of analysis and transferred directly into the injector of a
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HP5890II GC equipped with a DB-5 column (30 m, 0.32 mm inner diameter, 0.25 µm film
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thickness, J&W Scientific, Köln, Germany). Nitrogen (99.999% purity) served as carrier and
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make-up gas. Injection was done at a column temperature of 35 °C. After 5 min, the column
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temperature was increased to 60 °C at a rate of 5 °C min-1, then with 30 °C min-1 to 300 °C and a
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final hold of 10 min was included. Injector and detector temperatures were 240 and 320 °C,
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respectively.
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The chlorinated compounds were identified and quantified by injecting single compounds as standards with concentration between 5 and 100 µM.
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Determination of Bromide Concentrations. Bromide was quantified from 1 ml culture
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samples using a DIONEX – ISC2000 Ion Chromatography System equipped with an
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IonPacAS18/AG18 column (4 mm inner diameter, Thermo Fisher Scientific, Bremen, Germany).
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Resting Cell Activity Assay for Reductive Dehalogenases. Dehalogenase activity was
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analyzed based on a microtiter plate-format assay described previously,9 using whole cells as
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catalyst and reduced methyl viologen as an artificial electron donor. Used halogenated
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compounds are listed in Table S1 (Supporting Information). Final concentrations of 625 µM
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were added for each tested electron acceptor. Two hundred µl of assay solution were filled in
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each well and the reaction was started by adding 20 or 40 µl of cell suspension. Cells were 2-fold
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concentrated using a rotary evaporator before being added to the activity test. The microtiter 8
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plate was monitored with a microtiter plate reader within an anaerobic tent at 578 nm every 7.5
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minutes for 15 h at 30 °C. Each plate contained the samples with electron acceptor, negative
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controls without electron acceptor and a positive control with cells and 1,2,3-trichlorobenzene as
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electron acceptor, each in five parallels in addition to three parallel chemical controls with
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growth medium instead of the cells. The growth medium contained 50 µg L-1 vitamin B12. The
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specific activity was calculated from the slope of the linear part of the measured curves after
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correction using the negative controls. Protein amounts were calculated from cell numbers8,9
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using a value of 30 fg protein per cell.
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Calculation of Partial Charges. Different approaches have been described to compute atomic
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partial charges based on the electronic structure of a molecule. As atomic charges are no
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quantum chemical observables, there is no "canonical" way of obtaining them and all approaches
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have advantages and disadvantages. In this work, we used atomic charges based on the NPA
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(natural population analysis) method, which itself is based on the NBO (natural bond orbital)
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algorithm.29 In short, NBO constructs a unitary transformation of the canonical molecular
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orbitals (which are the result of the calculation of the electronic structure) into a set of spatially
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confined orbitals resembling the concept of chemical bonding. The NBOs are assigned to atoms
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and their occupation per atom is integrated, similar to the computation of Mulliken charges.13
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But in contrast to the latter, the NPA method is able to correctly handle a larger variety of bond
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types including those with a high degree of ionicity and features a higher numerical stability as
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well as a much lower dependence on basis set size.30 Mulliken and Hirshfeld partial charges were
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calculated additionally to test whether different charge models reproduce similar trends. The
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Mulliken charge of an atom is obtained by summing up the populations for all basis functions
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centered on the atom and subtracting them from the nuclear charge. Overlapping populations 9
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between two atoms are divided half in half therefore not taking into account the differences of
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electronegativity between two atoms. While the NBO and the Mulliken model are based on the
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analysis of wave functions the Hirshfeld model is a density-based population model. It makes
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use of a promolecule which is the sum of the ground-state atomic densities of the atoms of the
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real molecule. The electron density of the real molecule is partitioned onto the atoms of the
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promolecule corresponding to the ratio of the electron densities of the atoms of the promolecule.
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By subtracting the integrated density of an atom of the real molecule from the nuclear charge of
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the free atom of the promolecule the Hirshfeld atomic charge is obtained.
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The geometries of 58 molecules were optimized using Density Functional Theory level
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B3LYP/6-31G(d,p) in order to obtain a minimum energy structure for each molecule. Correct
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ground-state geometries for all compounds were confirmed by frequency analysis. All tasks
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including Mulliken,13 Hirshfeld31 (keyword IOP(6/79=1)) and NBO32 population analysis were
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performed using Gaussian 09 revision C.01.33
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To obtain NPA charges with solvation effect included, the CPCM solvation model34,35 was
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applied. Gaussian 09 CPCM calculations of the molecules were carried out with the two solvents
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chlorobenzene and water. Chlorobenzene was used to model the solvation within an enzyme
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pocket, where the effective permittivity is much lower than in water.36 The atomic radii required
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for CPCM were obtained by the UAKS method, as suggested in the literature.37 Additional non-
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electrostatic terms of the model were explicitly switched on ("dis", "rep", "cav"). Geometry
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optimizations with CPCM were carried out for all molecules to include the solvent effect on
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geometry.
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RESULTS
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Dehalogenation of homo- and heterocyclic aromatic compounds. Strain CBDB1 was
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cultivated with halogenated homo- and heterocyclic aromatic compounds as electron acceptor
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and hydrogen as electron donor. Reductive dehalogenation was observed for 2,3-dichloroaniline,
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2,3-, 2,5- and 2,6-dichlorobenzonitrile, 2-chloro-6-fluorobenzonitrile, 2,4,6-, 2,4- and 2,6-
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bromophenol, 4-bromo-3,5-dimethoxy- and 4-bromo-3,5-dihydroxybenzoic acid, 2,3- and 2,5-
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dichlorothiophene, 4,5- and 5-bromo-2-furoic acid, and 3-bromo-2-chloropyridine (Table 1,
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Figure 1 and 2). 2,4- and 2,6-dichloroaniline, and 2,3- and 2,6-dichloropyridine were not
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dehalogenated within a cultivation period of 140 days.
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First doses (10 µM to 100 µM or added as crystals, Supporting Information, Table S1) of 2,3-
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dichloroaniline, benzonitriles, halogenated phenols, thiophenes and 3-bromo-2-chloropyridine
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were dehalogenated within 70 days of cultivation. Additional doses of 50 µM 2,3-
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dichloroaniline, 40 - 60 µM 2,4-di-, 2,6-di- or 2,4,6-tribromophenol, 2,3- or 2,5-
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dichlorothiophene and 25 - 30 µM 3-bromo-2-chloropyridine were also dehalogenated (Figure 1
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and 2). Dehalogenation was detected after 7 days with bromophenols, after 18 days with 3-
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bromo-2-chloropyridine and after a lag phase of 10 to 30 days with 2,6-dichlorobenzonitrile or
234
2,3- and 2,5-dichlorobenzonitrile (data not shown). In cultures fed with 4,5-dibromo-2-furoic
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acid 400 µM bromide was released within 126 days (Figure 2). Dehalogenation of 800 µM 5-
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bromo-2-furoic
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dihydroxybenzoic acid was observed within 63 days of cultivation (Figure 2).
acid,
340
µM
4-bromo-3,5-dimethoxy-
and
410
µM
4-bromo-3,5-
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All tested halogenated compounds were stable in chemical controls without strain CBDB1
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except for 2,4,6-tribromophenol, 4,5-dibromo-2-furoic acid and 3-bromo-2-chloropyridine.
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These chemical controls contained growth medium instead of culture and therefore also 11
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contained vitamin B12 to account for corrinoid-mediated abiotic catalysis. In chemical controls
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with 45 µM 2,4,6-tribromophenol, 21.6 µM reacted abiotically within 70 days of incubation to
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2,4-dibromophenol whereas in parallel cultures with CBDB1 in the same time approximately 50
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µM of 2,4,6-tribromophenol were transformed to less brominated phenols and phenol. After 126
245
days of incubation, the chemical control with 4,5-dibromo-2-furoic acid contained about 50% of
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the bromide concentration found in cultures with strain CBDB1. 15 µM of 2-chloropyridine were
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released in chemical controls of 3-bromo-2-chloropyridine within 49 days, compared to 25.5 µM
248
in cultures inoculated with strain CBDB1 (Figure 2).
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Dehalogenation Pathways Catalyzed by Strain CBDB1. Dehalogenation pathways were
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determined to identify which halogen substituent was removed preferentially from a given
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compound (Table 1). The chlorine substituent in position 2 of 2,3-dichloroaniline was
252
reductively dechlorinated by strain CBDB1 and 3-chloroaniline was formed. The cyano-group of
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benzonitriles allowed the dechlorination of all ortho-chlorines. Substituents at other positions
254
and also the fluorine substituent in the ortho-position of 2-chloro-6-fluorobenzonitrile were not
255
removed. Strain CBDB1 fully dehalogenated all tested bromophenol congeners to phenol.
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Intermediates from 2,4,6-tribromophenol were 2,4-dibromo- and 4-bromophenol demonstrating
257
that the ortho-position was the preferred dehalogenation site. 2,4- and 2,6-dibromophenol were
258
debrominated to phenol via 4- and 2-bromophenol, respectively. 4-Bromo-3,5-dimethoxybenzoic
259
acid and 4-bromo-3,5-dihydroxybenzoic acid were both debrominated to the non-halogenated
260
3,5-dimethoxy- and 3,5-dihydroxybenzoic acid.
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In thiophenes, the position next to the sulfur heteroatom was the preferred dehalogenation site
262
(Figure 1c). 2,3-dichlorothiophene was only dehalogenated to 3-chlorothiophene. 2,5-
263
Dichlorothiophene, having both chlorine substituents flanked by the sulfur heteroatom, was fully 12
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dehalogenated to thiophene. 4,5-Dibromo- and 5-bromo-2-furoic acid were both biotically
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debrominated by strain CBDB1. The nitrogen heteroatom in pyridines did not support
266
dechlorination in ortho- or meta-position. In contrast, removal of bromine substituents in
267
pyridines was possible as shown with the transformation of 3-bromo-2-chloropyridine to 2-
268
chloropyridine.
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Growth of Strain CBDB1. To investigate if the different halogenated compounds were used
270
as an electron acceptor in organohalide respiration, strain CBDB1 was cultivated for at least two
271
transfers on compounds that were dehalogenated and cultures were monitored for growth and
272
dehalogenation. Transformation of 2,3-dichloroaniline, chlorinated benzonitriles, brominated
273
phenols, brominated dimethoxy- and dihydroxybenzoic acids, chlorinated thiophenes,
274
brominated furoic acids, and 3-bromo-2-chloropyridine was accompanied by an increase in cell
275
number of strain CBDB1. Initial cell numbers of 5x105 to 6x106 ml-1 and final cell numbers of
276
4x106 to 8x107 ml-1 were observed. An increase in cell numbers was not observed if no electron
277
acceptor was added. From the measured dehalogenation and growth data, molar growth yields of
278
0.1x1014 to 5x1014 cells per mol of halogen ion released were calculated (Table 1).
279
Resting Cell Activity Assay for Reductive Dehalogenases. In activity assays cell
280
suspensions of
strain CBDB1 cultivated with 1,2,3-trichlorobenzene dehalogenated 2,3-
281
dichlorobenzonitrile, all tested brominated phenols, 4-bromo-3,5-dimethoxybenzoic acid, 2,3-
282
dichlorothiophene and 2,5-dichlorothiophene (Table 1). The highest specific activities were
283
found for 2,4,6-tribromophenol (87.4 nkat mg-1), 2,3-dichlorobenzonitrile (50.6 nkat mg-1) and
284
2,3-dichlorothiophene (42.1 nkat mg-1). The lowest activity was determined for 2,5-
285
dichlorothiophene (4.5 nkat mg-1).
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Although dehalogenation of 2,3-dichloroaniline, 2,5-, 2,6-dichlorobenzonitrile, 2-chloro-6-
287
fluorobenzonitrile, 4-bromo-3,5-dihydroxybenzoic acid and 5-bromo-2-furoic acid was observed
288
during cultivation of strain CBDB1, no activity was measured in the enzyme activity assays.
289
These results were obtained with cultures grown with 1,2,3-trichlorobenzene as the electron
290
acceptor but also with cultures grown on the same electron acceptor that was later tested in the
291
activity test. All compounds except for 4,5-dibromo-2-furoic acid were stable in activity assays
292
as confirmed by chemical controls without cells.
293
Net Atomic Halogen Charges Predict Regioselectivity of Reductive Dehalogenation.
294
Partial charges were calculated for each atom in the tested halogenated anilines, benzonitriles,
295
benzoic acids, thiophenes, furoic acids and pyridines on the basis of the NBO (ܳ ேை ), Hirshfeld
296
(ܳ ௦ ) and Mulliken (ܳ ௨ ) models, separately (Table 2, Supporting Information, Tables S2
297
and S3). Additionally, electron densities were calculated for chlorobenzenes, chlorophenols and
298
selected bromobenzenes (Supporting Information, Tables S4-S7) and compared with previously
299
determined experimental data5,9,18 to analyze correlations between the occurrence of a
300
dehalogenation reaction and electron densities from a broader data basis. A total of 58 molecules
301
were analyzed.
302
All three partial charge models revealed for most of the analyzed compounds that
303
dehalogenation preferentially took place in the position having the most positive charge on the
304
halogen atom (ܳ ) and a more negative partial charge on the carbon atom (ܳ ). On the basis of
305
the most positive ܳ the Hirshfeld system predicted 96% of dehalogenation pathways correctly,
306
the Mulliken and NBO models 88%. Analyzing the most negative ܳ the Hirshfeld, Mulliken
307
and NBO models allowed for a correct prediction of dehalogenation pathways for 80%, 68% and
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76%, respectively. When calculating with phenolates, prediction rates on the basis of the most
309
positive ܳ using the Mulliken and NBO models were improved to 96% (data not shown).
310
While all models calculate the most positive partial charge at the halogen that was
311
preferentially dehalogenated, the NBO model reflected higher electronegativity of chlorine
312
compared to bromine and therefore predicted correctly the preferential removal of bromine from
313
2-chloro-3-bromopyridine.
314
Obtained charges on the halogen atom using the NBO model (ܳேை ) ranged from -0.312 for
315
the fluorine atom of 2-chloro-6-fluorobenzonitrile to +0.161 for the bromine atom in the ortho-
316
position of 4,5-dibromofuroic acid. Halogen substituents that were dehalogenated showed ܳேை
317
values between -0.094 (Cl2 of 2,5-dichlorophenolate) and +0.139 (5-bromofuroic acid).
318
The most negative ܳேை value was calculated for the C2 of 2,3-dichlorothiophene (-0.304) and
319
the most positive values for the C6 atom of 2-chloro-6-fluorobenzonitrile (+0.484) and the C2/6
320
atoms of 2,6-dichloropyridine (+0.198). ܳேை values of carbon atoms associated with
321
substituents that were dehalogenated were between -0.304 for the C2 carbon atom of 2,3-
322
dichlorothiophene and -0.009 for the chlorine substituted carbons of 2,6-dichlorobenzonitrile.
323
Calculated specific enzyme activities (Table 1) were compared to ܳேை values of the
324
respective compound. When comparing within one compound class, higher (i.e. more positive)
325
ܳேை values were paralleled with higher enzyme activity.
326
The NBO model permitted the calculation of σ and π electron distributions. For halogenated
327
anilines, benzonitriles, benzoic acids, thiophenes, furoic acids and pyridines experimentally
328
tested in this study and chlorobenzenes, chlorophenols and selected bromobenzenes from
329
previous studies σ and π electron distributions were modelled based on NBO partial charges
330
reflecting inductive and mesomeric effects, respectively (Supporting Information, Table S8-S10). 15
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88% of the regioselective dehalogenation reactions catalyzed by strain CBDB1 were predicted
332
correctly when using the halogen atom with the most positive σ partial charge as predictor. In
333
contrast, only 64% of the reactions were correctly predicted when using the halogen with the
334
most positive π electron partial charge.
335
To estimate is the influence of solvation on electron densities within the halogenated electron
336
acceptors NBO charges for halogen and carbon atoms (ܳேை and ܳேை ) were calculated for the
337
molecules listed in Table 1 with the solvents water and chlorobenzenes (Supporting Information,
338
Table S11 and S12, respectively). The results demonstrate that solvation did not change the
339
relative values of partial changes within a given molecule to each other.
340
DISCUSSION
341
Dehalococcoides mccartyi strain CBDB1 transforms a much wider range of halogenated
342
aromatics than previously anticipated including aromatics substituted with cyanide, amino,
343
hydroxyl or methoxyl groups and heterocycles with oxygen, sulfur or nitrogen heteroatoms.
344
These functional groups are present in many commercially used halogenated pesticides,
345
pharmaceuticals and flame retardants. We here demonstrate that non-halogen substituents or
346
heteroatoms can promote or inhibit dehalogenation reactions by their influence on the electron
347
density distribution within the molecule. Most of the investigated molecules were used by strain
348
CBDB1 as an electron acceptor for energy conservation via organohalide respiration allowing
349
effective transformation in bioremediation. Growth yields between 0.9 x 1013 and 5.1 x 1014 cells
350
per mol of halogen released were similar to previous findings with other electron acceptors.5,9
351
Growth yields were generally lower for electron acceptors which reacted also abiotically or
352
which contained phenolic groups. For abiotically reacting molecules, dehalogenation might be 16
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partially uncoupled from growth. Compounds containing phenolic groups inhibit growth by
354
interfering with the proton membrane gradient.5,38
355
The systematic investigation of functional group influence onto reductive dehalogenation may
356
allow for an improved reactivity prediction of newly introduced halogenated aromatics. So far,
357
predictions of dehalogenation pathways were built exclusively on known dehalogenation
358
pathways which did not allow extrapolation onto unknown aryl halides. Therefore, we here
359
studied why and how halogen positions and additional substituents are relevant to reductive
360
dehalogenation.
361
Quantum chemical analysis of aromatic halides ArX across several compound classes using
362
three different partial charge models unravels that the positive partial charge required for
363
reduction of ArX is located on a halogen and not on a carbon atom. In addition the analysis
364
shows that the net halogen charge (ܳ ) can serve as a good predictor of the regioselectivity
365
observed for the reductive dehalogenation by D. mccartyi strain CBDB1. For compounds of
366
which two or more reaction products were formed concurrently the ܳ of simultaneously
367
removed halogens were relatively close to each other (Supporting Information, Table S4 and S6).
368
This is consistent with a previous study in which a correlation between Mulliken charges and
369
reductive dehalogenation was found for chlorinated benzenes, phenols and dioxins.11,39
370
When a molecule was substituted with only one type of halogen, all models correctly predicted
371
the dehalogenation pathway when using the most positive halogen net atomic charge. The
372
Mulliken and the Hirshfeld partial charge model however, were not suitable for the prediction of
373
dehalogenation pathways when a molecules contained both, bromine and chlorine substituents.
374
Also they did not give correct predictions when comparing compounds that only differed by the
375
type of the halogen substituents (e.g. brominated vs. chlorinated benzenes). Then, both charge 17
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376
models assigned more negative partial charges to bromine substituents compared to chlorine
377
substituents (Supporting Information, Table S2), thus not reflecting the higher electronegativity
378
of chlorine compared to bromine.
379
Although all partial charge models showed good prediction rates for dehalogenation pathways
380
when using the halogen atomic net charge, each model showed few inconsistencies with
381
experimentally identified dehalogenation pathways. This emphasizes the necessity for the
382
applications of several charge models in parallel for a verification of obtained values.
383
A threshold for reductive dehalogenation according to a specific partial charge value was not
384
identified when analyzing all compound classes together. Possibly, a threshold for reductive
385
dehalogenation can be identified when analyzing more congeners of only one compound class or
386
by including further parameters into the charge calculation. Similarly, partial charges showed no
387
correlation with specific activities when analyzing all compounds together. However, when
388
considering only one compound class less negative halogen atomic net charges were paralleled
389
with higher specific activity.
390
In general, ܳ values were more positive for aromatics with a higher number of halogen
391
substituents, which reflects the observed preferential transformation of highly halogenated
392
compounds (Supporting Information, Tables S2, S4 and S6, for experimentally tested substrates,
393
halogenated benzenes and halogenated phenols, respectively). The values of the net atomic
394
carbon charge performs significantly inferior as a predictor for regioselectivity, but a trend
395
towards more negative ܳ values indicating enhanced reductive dehalogenation was observed.
396
However, a rough negative correlation between carbon and halogen charges was expected due to
397
a polarization of the carbon halogen bond caused by their difference in electronegativity.
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According to our modelled ܳேை values the cyano-group of benzonitrile induced a more
399
positive charge on the ortho halogen atom than the hydroxyl- and amino-group in 2,6-
400
dichlorophenol and 2,6-dichloroaniline (Table 2). This is in line with the experimental data
401
showing that dehalogenation was most extensive for 2,6-dichlorobenzonitrile and least extensive
402
for chlorinated anilines, when comparing chlorinated benzonitriles, benzenes, phenols and
403
anilines5,8,18. This roughly corresponds with Hammett constants placing the electron withdrawing
404
effect in the order -CN > -X > -OH> -NH2.40 Hence, substituents withdrawing electron density
405
from the halogen enhanced reductive dehalogenation. When density functional theory modelling
406
is not available, Hammett constants might serve as a rule of thumb to estimate the enhancing
407
effect of substituents onto reductive dehalogenation.
408
When investigating reductive dehalogenation of halogenated heteroaromatic compounds with
409
strain CBDB1, electron-rich heteroaromatic compounds such as chlorinated thiophenes were
410
dehalogenated, whereas electron-poor chlorinated pyridines were not dehalogenated. Brominated
411
furoic acids were dehalogenated to non-halogenated reaction products. Carbon atoms flanking
412
the sulfur or oxygen heteroatoms had more negative net carbon charges than carbon atoms
413
flanking the nitrogen heteroatom of chlorinated pyridines. Nitrogen-heteroatom flanking carbon
414
atoms showed one of the most positive partial charges of all analyzed compounds (Table 2).
415
For different halogen substituents an enhanced removal in the order -Br > -Cl > -F was
416
observed, i.e. fluorine was not removed at all, and bromine substituents were removed to a
417
further extent than chlorine substituents, when comparing for instance 2,3-dichloropyridine with
418
2-chloro-3-bromopyridine, 2,6-dichlorobenzonitrile with 2-chloro-6-fluorobenzonitrile, or other
419
brominated with chlorinated molecules tested in the current or in previous studies.9,21 These
420
findings are reflected in the NBO atomic net charges of halogens, which have a more positive 19
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421
partial charge for bromine compared to chlorine and for chlorine compared to fluorine in
422
comparable molecules. Also they are in line with reduction potentials reported for
423
monohalogenated benzenes yielding the order Br > Cl > F with Br having the least negative
424
reduction potential.41
425
Previous models suggested a SNAr reaction as mechanistic model for reductive
426
dehalogenation. An enhanced removal of halogens in the order -Br > -Cl > -F, is however in
427
contrast with the decrease of CAr-X bond dissociation energy in the order F > Cl > Br > I,42
428
reflecting the trend of the halogen facility as a leaving group as opposed to the halogen polarity
429
(inductive) effect that governs the readiness for SNAr reactions (Figure 3a). The increase in SNAr
430
reactivity with increasing electron deficiency at the aromatic ipso carbon as site of nucleophilic
431
attack is also not compatible with our present finding that the halogen and not the carbon with
432
the most positive net atomic charge predicts the regioselective dehalogenation pathway.
433
Similarly, observations made for heteroaromatic compounds contradict previous reaction models
434
based on a SNAr mechanism which would predict enhanced removal for halogens substituted to
435
carbons with a more positive partial charge as it was observed for in pyridines.
436
A more realistic option for the underlying reaction mechanisms is a dissociative single electron
437
transfer process (SET)43-45 (Figure 3b) in which initial electron transfer from B12CoI to the halide
438
could proceed in a concerted manner (SET, path 1) or in a consecutive manner involving a
439
radical anion ArX −• (SET, path 2). The generated radical Ar • yields after recombination with
440
B12CoII and protonation the reduced product ArH. If the initial electron transfer step is rate-
441
determining, the reaction rate is expected to correlate with the reduction potential E0
442
(ArX/ArX− •) within a given compound class. Heavy metals M including cobalt with their d8
443
configuration are known for their readiness to form hydrogen bonds as Lewis bases providing 20
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support for an initial single electron transfer from the corrinoid cobalt to the halogenated
445
substrate. Experimental findings suggest that the hydrogen bond to a metal atom as Lewis base
446
may take place intermediately on the way of a proton transfer to the metal, and thus can also be
447
understood as an arrested protonation. The d8 electron configuration facilitates a stabilizing 3-
448
center-4-electron interaction M···H–Y (Y = H-bond donor atom) with a partial d σHY*
449
electron transfer into the respective antibonding orbital of the Lewis acid HY. The readiness of
450
d8 metals to accept H bonds reflects their electron donor capability.
451
The phenomenon of halogen bonding X···B between a halogen atom X acting as Lewis acid
452
(electron acceptor) through its apical σ-hole and a Lewis base B, suggests that the latter may also
453
be formed with d8 heavy metals such as CoI as electron donor. The ability of B12CoI as H bond
454
acceptor has been demonstrated through computational chemistry, confirming the affinity of its
455
doubly occupied dz2 orbital for attractive interactions with Lewis acids. For Ni0 with a d8 electron
456
configuration as present in CoI, an electron transfer step through a metal attack at the halogen
457
site of X–C has been considered as one of several mechanistic options. An involvement of the
458
apical halogen σ−hole of X-CAr was indirectly supported by the improved predictions on the
459
basis of positive partial σ charges observed in this study. Electron transfer from the dz2 orbital of
460
B12CoI to the apical σ-hole X site of X–CAr driven by the attractive interaction of an emerging
461
Co-halogen bond B12CoI···X–CAr would initially populate the σCX* antibonding orbital (Figure
462
4), leading to a transient σ* radical anion [B12CoII···X–···Ar]• with a substantially weakened bond
463
between CAr and X. Now, the complex is prepared for a concerted dissociation into its
464
components B12CoII, X– and Ar• (Figure 3b, path 1). The neutral radical Ar• is relatively stable
465
due to delocalization of the unpaired electron through its aromatic π system, and together with
466
the thermodynamically stable halide X– pushes the dissociative electron transfer step to the 21
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467
product side disfavoring the formation of a radical anion intermediate ArX− •. Yet, it is not clear
468
why electron-poor pyridines did not invoke organohalide respiration, which would be expected
469
to be more reactive in both SNAr (because of the more positively polarized ipso CAr) and SET
470
mechanism (because of a generally larger electron affinity of the molecule). However, our
471
findings highlight the important role of the halogen-carbon bond polarization for the molecule
472
reactivity with Co(I) of reductive dehalogenases.
473
Local reactivity parameters such as the site-specific electrophilicity46 may provide additional
474
insight into the halogen affinity for its role as SET electron acceptor. Also, hydrogen bonds
475
between substrate and the protein environment in the active site of the enzyme will influence
476
substrate reactivity as suggested recently for the binding of ortho-halogenated phenols in the
477
soluble and oxygen-tolerant enzyme of Nitratireductor pacificus (NprdhA).27 Studying the
478
crystal structure of respiratory membrane-bound dehalogenases and their non-phenolic substrates
479
could give a more general picture of the B12-organohalogen interaction. In addition, reaction
480
barriers associated with different mechanisms and for different ligands could be characterized
481
through quantum chemical analyses of the B12CoI catalysis process, following approaches
482
successfully undertaken in the area of P450 catalysis.47,48 The very small active site in the
483
recently described three-dimensional structure of the RdhA protein of Sulfurospirillum
484
multivorans corroborates the proposed initial attack on the halogen atom.26 Similarly, modeling
485
of a NprdhA interaction with its substrate 3,5-dibromo-4-hydroxy benzoic acid suggested a
486
localization of the halogen directly above the cobalt.27 The presently introduced data obtained
487
with growing, organohalide-respiring anaerobic bacteria and electron density modelling together
488
with the characterization of the two reductive dehalogenase structures and EPR spectra26,27
489
strongly suggest the halogen as the primary site of attack for Co(I). These findings can be 22
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explained by the electronic structure characteristics of the d8 Co(I) supernucleophile, the affinity
491
of d8 heavy metals for hydrogen bonding49 and an apical σ-hole of halogens.
492
D. mccartyi strain CBDB1 is specialized on reductive dehalogenation of halogenated aromatic
493
compounds. The combination of microbiology, computational modeling and enzymatic studies
494
revealed the influence of the electron acceptor’s chemical properties on reductive
495
dehalogenation. Functional groups withdrawing electron density from the halogen enhance
496
reductive dehalogenation. Although other D. mccartyi strains such as strains DCMB510 and 19550
497
catalyze variations of the dehalogenation pathways catalyzed by strain CBDB1 functional groups
498
of the electron acceptors withdrawing electron density from the halogen still appear to have
499
major impact on the pathways. It is undisputed that the three-dimensional conformation of the
500
active site in the enzyme has pivotal influence on the regioselectivity of the catalyzed reaction.
501
Further systematic investigation using model organisms from different dehalogenating bacterial
502
groups will have to explore the influence attributable to the electron distribution within the
503
electron acceptor in comparison with the influence attributable to the structure of the catalyzing
504
enzyme. Together our findings give evidence that B12-halogen interactions take place in
505
anaerobic organohalide respiration and allow for an improved fate prediction of halogenated
506
aromatics in microbially habituated anaerobic environments.
507
AUTHOR INFORMATION
508
Corresponding Author
509 510 511
*Phone: 49-341-235-1435; e-mail:
[email protected] Present Addresses ǂ
Technische Universität Berlin, Bioprocess Engineering, Ackerstr. 76, 13355 Berlin, Germany 23
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512 513
514
Page 24 of 37
Notes The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
515
The authors thank Ulrich Wagner for critical discussion and helpful comments, Bernd Krostitz
516
for technical support. The work was supported by the ERC (project Microflex), DFG (FOR1530)
517
to L.A. and UFZ IP CCF to L.A. and G.S.
518 519
ASSOCIATE CONTENT
520
Supporting Information Available
521
Table S1: Used electron acceptors; Tables S2 to S10: Data of electron density calculation
522
according to the NBO, Hirshfeld and NBO models for different halogenated compounds on
523
halogen and carbon atoms. This information is available free of charge via the Internet at
524
http://pubs.acs.org.
525
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FIGURE LEGENDS
527 528
Figure 1. Transformation of halogenated aromatics in cultures of strain CBDB1. (a) Anilines (○
529
- 2,3-dichloroaniline, ● - 2,4-dichloroaniline; - 2,6-dichloroaniline), (b) benzonitriles (○ - 2,3-
530
dichlorobenzonitrile, ● - 2,5-dichlorobenzonitrile, - 2,6-dichlorobenzonitrile, □ - 2-chloro-6-
531
fluorobenzonitrile), (c) thiophenes (○ - 2,3-dichlorothiophene, ● - 2,5-dichlorohiophene) and (d)
532
pyridines (○ - 2,3-dichloropyridine; ● - 2,6- dichloropyridine). Shown are mean values and
533
standard deviations of two to three parallel cultures. Negative controls without inoculum or with
534
killed inoculum showed no decrease of electron acceptor concentrations. Arrows indicate a re-
535
feeding with electron acceptors 2,3-dichloroaniline (a), 2,3- and 2,5-dichlorothiophene (c).
536 537
Figure 2. Dehalogenation of brominated compounds in cultures of strain CBDB1. (a) Bromide
538
release from 4,5-dibromo-2-furoic acid (4,5-BDrFa) after 126 days of incubation and from 5-
539
bromo-2-furoic acid (5-BrFa), 4-bromo-3,5-dimethoxybenzoic acid (BrDMeOBa) or 4-bromo-
540
3,5-dihydroxybenzoic acid (BrDOHBa) after 63 days of incubation. (b) Formation of phenol
541
from 2,4-dibromophenol (○), 2,6-dibromophenol (▼) or 2,4,6-tribromophenol (●). (c)
542
Transformation of 3-bromo-2-chloropyridine in CBDB1 cultures (■) and in abiotic controls (□,
543
dashed lines). The product 2-chloropyridine accumulated much more in CBDB1 cultures (●)
544
than in chemical controls (○, dashed lines). Arrows indicate re-feeding of CBDB1 cultures with
545
the electron acceptor 3-bromo-2-chloropyridine. Shown are mean values and standard deviations
546
of two parallel cultures of a second transfer.
547
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548
Figure 3. Possible reaction mechanisms for the reactive dechlorination of substituted aromatics
549
by strain CBDB1. (a) SNAr, nucleophilic aromatic substitution; (b) SET, single electron transfer.
550 551
Figure 4. Single electron transfer model. In the initial step one electron is transferred from the
552
doubly occupied dz² orbital of CoI (d8) to the empty antibonding C-X σ* orbital at the backside of
553
the halogen atom attached to aromatic carbon.
554
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TABLES
556 557 558
Table 1. Dehalogenation reactions catalyzed by strain CBDB1 and molar growth yields when grown on different halogenated compounds and specific dehalogenating activity of resting cells previously grown on 1,2,3-trichlorobenzene. Molar growth yield Electron acceptor
Dehalogenation product(s)
(cells/mol halogen released)
Sp. Act.
(g protein/mol halogen released)
(nkat/mg of protein)
1.7-2.6
n.d.
Aromatic compounds with non-halogenated substituents 2,3-dichloroaniline
→ 3-chloroaniline
5.7 - 8.7 x 1013
2,4-dichloroaniline
not dehalogenated
no growth
n.d.
2,6-dichloroaniline
not dehalogenated
no growth
n.d.
2,3-dichlorobenzonitrile
→ 3-chlorobenzonitrile
2.3 x 1014
6.9
50.6 ± 4.5
2,5-dichlorobenzonitrile
→ 3-chlorobenzonitrile
5.1 x 10
14
15.3
n.d.
2,6-dichlorobenzonitrile
→ 2-chlorobenzonitrile → benzonitrile
3.5 x 1014
10.5
n.d.
2-chloro-6-fluorobenzonitrile
→ 2-fluorobenzonitrile1
2.3 x 1014
6.9
n.d.
2,4,6-tribromophenol
→ 2,4-dibromophenol → 4-bromophenol → phenol
3.7 x 1013
1.1
87.4 ± 3.7
2,4-dibromophenol
→ 4-bromophenol → phenol
3.0 x 1013
0.9
41.7 ± 3.9
2,6-dibromophenol
→ 2-bromophenol → phenol
3.7 x 10
13
1.1
14.3 ± 1.9
4-bromo-3,5dimethoxybenzoic acid
→ 3,5-dimethoxybenzoic acid
1.8 x 1014
5.4
34.0 ± 4.7
4-bromo-3,5dihydroxybenzoic acid
→ 3,5-dihydroxybenzoic acid
4.4 x 1013
1.3
n.d.
Heterocyclic compounds
559 560
2,3-dichlorothiophene
→ 3-chlorothiophene
9.9 x 1013
3.0
42.1 ± 1.3
2,5-dichlorothiophene
→ 2-chlorothiophene → thiophene
9.3 x 1013
2.8
4.5 ± 1.1
4,5-dibromo-2-furoic acid
bromide released, product not determined
0.9 x 1013
0.3
a.r.
5-bromo-2-furoic acid
→ furoic acid
9.5 x 1013
2.9
n.d.
2,3-dichloropyridine
not dehalogenated
no growth
n.d.
2,6-dichloropyridine
not dehalogenated
no growth
n.d.
3-bromo-2-chloropyridine
→ 2-chloropyridine
9.4 x 1013
2.8
30.2 ± 1.7
-
n.d. – no activity detected; a.r. – abiotic reaction, but Br release was higher in biotic reactions 1 A product was detected that was not 2-chlorobenzonitrile, presumably 2-fluorobenzonitrile. 27
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561
Table 2 ܳேை and ܳேை values for homo- and heteroaromatic compounds tested experimentally
562
in this study. Indicated in bold are positions from which halogen substituents were removed by
563
strain CBDB1. Grey background highlights halogen substituents with most positive ܳ or
564
ܳ values. C2 ܳேை
C3 ܳேை
ܳேை
C4 ܳேை
ܳேை
C5 ܳேை
ܳேை
C6 ܳேை
ܳேை
ܳேை
Aromatic compounds with non-halogenated substituents 2,3-dichloroaniline
0.008
-0.110 0.019
-0.049
2,4-dichloroaniline
-0.013 -0.074
2,6-dichloroaniline
-0.015 -0.075
2,3-dichlorobenzonitrile
0.070
-0.035 0.043
2,5-dichlorobenzonitrile
0.047
-0.017
2,6-dichlorobenzonitrile
0.050
-0.001
2-chlorobenzonitrile
0.038
-0.010
2-chloro-6-fluorobenzonitrile
0.048
-0.002
2,4,6-tribromophenol
0.072
-0.162
0.077
-0.123
2,4-dibromophenol
0.089
-0.156
0.063
-0.130
2,6-dibromophenol
0.057
-0.169
2-bromophenol
0.073
-0.165
-0.007 -0.069 -0.015 -0.075 -0.067 0.023
-0.052 0.050
-0.001
-0.312 0.484
4-bromophenol
0.049
-0.138
4-bromo-3,5-dimethoxybenzoic acid
0.106
-0.182
4-bromo-3,5-dihydroxybenzoic acid
0.083
-0.221
0.102
-0.147
0.088
-0.156
0.013
0.198
Heterocyclic compounds 2,3-dichlorothiophene
0.071
-0.305 0.037
2,5-dichlorothiophene
0.055
-0.285
2-chlorothiophene
0.046
-0.287
-0.124 0.055
4,5-dibromo-2-furoic acid
0.127
5-bromo-2-furoic acid 2,3-dichloropyridine
0.026
0.174
2,6-dichloropyridine
0.013
0.198
3-bromo-2-chloropyridine
0.025
0.175
0.039
0.107
-0.285
-0.249 0.161
0.182
0.139
0.187
-0.106
-0.177
28
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