Environmental Toxicology and Chemistry, Vol. 19, No. 3, pp. 543±548, 2000 ᭧ 2000 SETAC Printed in the USA 0730-7268/00 $9.00 ϩ .00
DECHLORINATION OF SUBSTITUTED TRICHLOROMETHANES BY AN IRON(II) PORPHYRIN
RICHARD A. LARSON* and JAVIERA CERVINI-SILVA Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, 1101 West Peabody Drive, Urbana, Illinois 61801, USA
(Received 24 February 1999; Accepted 22 June 1999)
AbstractÐIron(II) porphyrins complex with many organochlorine compounds and oxidize by dechlorinating them. For a series of Ϫ trichloromethyl derivatives, CCl3R(Rϭ NO2, CHCl2, CN, Cl, COO ,CH3, H, C(O)NH2, and CH2OH), heme oxidation occurred in two consecutive steps: heme-CCl3R complexation, and (usually slower) inner-sphere electron transfer. Stability of the intermediate • CCl2R radical, which is strongly correlated with the ability of the R group to delocalize electron density, governed the overall electron transfer rate.
KeywordsÐTrichloromethanes Inner-sphere electron transfer Group electronegativity
INTRODUCTION solution [5±7]. In this study, we examine the reactivity of an Chlorinated organic compounds have very different sus- iron(II) complex, heme, toward a series of chlorinated com- ceptibilities toward reduction [1±3]. Iron(II) acts as an elec- pounds, CCl3R, possessing different R electronic properties. tron-transfer mediator either at mineral surfaces or in solution. We use linear free-energy relationships to help explain our Metallic iron surfaces contain regions including iron(II) and observations. iron(III) minerals as well as iron(0), but the chemical impor- MATERIALS AND METHODS tance of the various species is not fully understood [4,5]. Previous studies have examined reactions of the biologi- Materials cally and environmentally important iron(II) porphyrins with Pentachloroethane (98%), carbon tetrachloride (99%), chlo- chlorinated methanes [6±9] and halogenated ethanes [8±12]. roform (99%), trichloroacetonitrile (99%), 1,1,1-trichloroeth- Castro et al. [6,7] proposed that dechlorination of alkyl halides ane (99%), 2,2,2-trichloroacetamide (99%), 2,2,2-trichloro- (RX where X ϭ Cl) occurred via abstraction of a chlorine ethanol (99%), hematin (ϩ99%; Fig. 1), and sodium dithionite atom by the Fe(II) center to form an Fe(III)-Cl complex and (98%) were purchased from Aldrich Chemical Company (Mil- a free radical according to inner-sphere electron transfer: waukee, WI, USA). Trichloroacetic acid (99%), sodium bi- slow carbonate, and sodium carbonate were purchased from Fisher FeIIϩ RX ↔ Fe II (RX) → [Fe´´X´´R]± (Fair Lawn, NJ, USA). Trichloronitromethane (98%) was pur- → FeIII X ϩ R• (slow) (1) chased from Mallinckrodt (Paris, KY, USA), industrial ethanol (USP) from McCormick Distilling Company (Weston, MO, FeII ϩ R• → FeII (R• ) ϩ Hϩ → FeIII ϩ RH (fast) (2) USA), and pentane for THM analysis HP; from Burdick and In contrast, Perlinger et al. [10] studied dehalogenation of Jackson (Muskegon, MI, USA). polyhalomethanes and ethanes (including CCl3R with R ϭ H, Hematin stock solution Cl, CHCl2, CCl3, and CH3) in the presence of an iron(II) por- phyrin (meso-tetrakis[N-methylpyridyl] iron porphyrin) and A procedure modi®ed from that reported elsewhere ] was cysteine, and they suggested an outer-sphere electron transfer, used to prepare the stock solution of hematin, an iron(III) in which the ®rst step was an electron transfer to the halo- porphyrin. Hematin (10 mg, 1.6 ϫ 10Ϫ5 mol) was added to Ϫ3 genated alkanes (R Ϫ X) followed by either formation of a 100 ml of 1:1 v/v aqueous NaHCO3 (5.2 ϫ 10 M; pH, 8): carbanion [R Ϫ X]Ϫ• (Eqn. 3), dissociation of the weakest ethanol and stirred for 4 h. The green solution was then de- carbon-halogen bond (Eqn. 4), or both: canted from the insoluble residue; its concentration was 1 ϫ Ϫ4 R Ϫ X ϩ eϪϪ↔ [R Ϫ X] ••→ R ϩ XϪ (3) 10 M. Later, it was purged with argon (99.9%) for 15 min and used to prepare a solution of heme (Fig. 1). R Ϫ X ϩ eϪ → R• ϩ XϪ (4) Heme preparation To our knowledge, however, little attention has been paid to how the electronic properties of substituents in chlorinated From the stock solution, a 5-ml aliquot of hematin was compounds affect the mechanisms of their reaction toward added to 0.02 g (1.15 ϫ 10Ϫ4 mol) of solid sodium dithionite iron(II) in solution. Molecular structure may be important for (Na2S2O4) in a 6-ml, oxygen-free vessel and shaken manually. predicting their rates of reduction by zero-valent iron [1,2], Dithionite serves as a source of reducing sulfoxyl radical an- Ϫ iron(II)-containing clays [3] and minerals [4], and iron(II) in ions, ´SO2 , which forms when the salt is dissolved in water at alkaline pHs [13]. A rapid change of color from dark green * To whom correspondence may be addressed ([email protected]). (hematin) to bright red (heme, the iron[II] form) indicated the
543 544 Environ. Toxicol. Chem. 19, 2000 R.A. Larson and J. Cervini-Silva
Fig. 2. Spectra obtained during determination of chlorohemin for- mation from the reaction between heme and trichloromethanes. Fig. 1. Molecular structure of hematin (a), heme (b), and chlorohemin (c). nitrile, methanol, and distilled water (50:25:25) were used as
reference to estimate RF values, which corresponded to 0.30 change in oxidation state (solution A). Concentrations of he- and 0.38, respectively. Two major spots (RF ϭ 0.31 and 0.37, matin (ϭ575 nm, ⑀ϭ34.4 L/mol cm [6,7,14]) and heme respectively) were observed after the elution of the reaction (ϭ566 nm, ⑀ϭ29.1 L/mol cm) were determined spectro- mixture (discussed previously) and suggested the formation of scopically. both chlorohemin and hematin.
Reaction of trichloromethanes and heme RESULTS AND DISCUSSION Solutions of CCl R (solution B) were prepared in 1:1 v/v 3 The oxidation of heme in the presence of CCl3R(Rϭ NO2, water:ethanol. The vessels were shaken manually until no Ϫ CHCl2, CN, Cl, COO ,CH3, H, C[O]NH2, and CH2OH) was emulsion was visibly detected (®nal concentration, 0.01 M). correlated with the decrease in absorbance of the signal cor- Solution A (1.5 ml), solution B (0.5 ml), and 1:1 v/v bi- responding to the iron(II) form (ϭ566 nm) coupled with carbonate-ethanol (1.0 ml) were added to a screw-capped cu- buildup of the iron(III) form. We con®rmed the formation of vette to adjust the concentration of sodium bicarbonate to 4.5 chlorohemin, an iron(III) porphyrin bearing a chlorine atom Ϫ3 Ϫ3 ϫ 10 M, CCl3Rto1.7ϫ 10 M, and heme to approximately as a ligand, by its absorption at a of 490 and 615 nm. During 5 ϫ 10Ϫ5 M, respectively. The rate of heme loss was deter- the reaction, a smooth change occurred isosbestically, from mined using a Beckman 7400 DU diode-array spectrometer the heme to the chlorohemin spectrum (Fig. 2), which was (Beckman, Fullerton, CA,USA). consistent with the formation of chlorohemin (ϭ490 and 605nm [6,7]) and not with the formation of a stable heme- Chlorohemin identi®cation CCl3R complex (ϭ470 nm [9]) reported elsewhere. The Hematin (ϭ575 nm) and chlorohemin (ϭ490 and 615 formation of chlorohemin from heme was crosschecked using nm, ⑀ϭ34 L/mol cm [15]) were differentiated by ultraviolet thin-layer chromatography; in these experimental conditions, spectroscopy and thin-layer chromatography. The elution sol- RF values were comparable in magnitude that those reported vent consisted of the mixture of two solutions prepared sep- elsewhere [16]. arately. Solution 1 was prepared by adding cetyltrimethylam- The shift of absorbance from 566 to 600 nm in the presence monium bromide (3%) in water, acetonitrile, and methanol in of chloropicrin (CCl3NO2) and trichloroacetonitrile (CCl3CN) a ration of 55:3:42 v/v/v; solution 2 was 0.02 M aqueous HCl was found to be extremely fast, and in these cases, the rates prepared just before use with reverse osmosis-distilled water. of heme loss were extrapolated from an additional study of
Five milliliters of solution 1 and 2 ml of solution 2 were mixed, the loss rate as a function of [RCCl3]0. Products of CCl3NO2 placed in a 150-ml closed beaker, and equilibrated for 30 min loss were not determined (either one or two chlorine atoms before introduction of the plate. Samples of hematin stock could have been displaced). Data for CCl4, however, suggested solution and the reaction mixture were spotted on a 8- ϫ 4- that one chlorine at a time was removed. The rate of dechlo- cm sheet of Whatman ¯exible plates for thin-layer chroma- rination of CHCl3 was much slower than that of CCl4. tography (layer, 250 m) coated with silica gel 1.2 cm above In most experiments where chlorohemin was formed, stoi- the lower edge. The spots were dried with argon before elution chiometric heme loss was faster than chlorohemin formation, of the plates. The elution time was 20 min, and the plates were suggesting that CCl3R-heme complexation occurs at a com- revealed in a 150-ml covered beaker containing 30% NH3. parable rate to that of electron transfer (Fig. 2) and that the Samples of hematin and chlorohemin in a mixture of aceto- heme-mass balance may be described as Dechlorination of substituted trichloromethanes Environ. Toxicol. Chem. 19, 2000 545
ation rate, and kii to the loss of the precursor complex and heme oxidation. If the concentration of the precursor complex that may form is limited by the amount of heme in solution (Eqn. 8), the
initial concentration of CCl3R is the same in all the experi-
ments. Thus, ki[CCl3R] may be written as kiЈ, and in the case
where kii k kϪi, Equation 9 simpli®es to Equation 11:
[heme] Ն [heme ´ ´ CCl3 R] (10) Ϫd[heme] d[heme ´ ´ CCl R] ϭϭ3 (kЈϪk )[heme] (11) dt dt iii Fig. 3. Pro®les of heme loss in the presence of trichloromethanes. where heme ´ ´ CCl R is proportional to the amount of total Ϫ 3 Circles refer to the trend observed when R ϭ NO2, CHCl2, Cl, COO , CH , and H (pro®le I). Squares refer to the trend when R ϭ CN heme, [heme]T. Thus, Equation 11 may also be written as 3 Equation 13: (pro®le II). Triangles refer to the trend when R ϭ CONH2 or CH2OH (pro®le III). [heme ´ ´ CCl3 R] ϰ [heme] (12) Ϫd[heme] ϰ [heme](kiiiЈϪk ) (13) [heme]Tϭ [heme] freeϩ [heme] complexed and (5) dt
[heme]TϪ [heme] freeϭ [heme] complexed (6) The rate of heme oxidation depends on the heme and CCl3R concentrations, and it is limited by the stability of the precursor where [heme]Trefers to the total heme present in solution complex (Eqn. 13). The lifetime of the precursor complex () formed during hematin reduction, [heme]complexed to complexed Ϫ1 [17,18] orϭkiiiЈϪk , varies from one CCl3R compound heme, and [heme]free(??) to uncomplexed heme. If [heme]free K to another and depends on the molecular structure. Pro®le I [heme]complexed, then the heme rate law expression may be ap- Ϫ (R ϭ NO2, CHCl2, Cl, COO ,CH3, H) illustrates the case proximated as Ϫ1 where electron transfer is the rate-limiting step and ഠ kii. In pro®le II (R ϭ CN), complexation is the rate-limiting step, d[heme]T d[heme]complex ഠ (7) andϪ1 k Ј . Pro®le II may be considered to be a particular dt dt ഠ i case of pro®le I, in which the nitrile group and chlorine atoms We identi®ed three apparently different courses of reaction may compete to complex with iron(II) center via a lone-pair between CCl3R and heme (Fig. 3). For most of the compounds interaction (Fe:NϵC and Fe:Cl, respectively) and retard the Ϫ (R ϭ NO2, CHCl2, Cl, COO ,CH3, H; pro®le I), heme con- electron transfer [19]. Pro®le III (R ϭ CONH2,CH2OH) cor- centration decreased rapidly and then remained constant over responds to the case in which the concentration of heme re- time. In the presence of trichloroacetonitrile (R ϭ CN), how- mained relatively constant, no electron transfer was observed, Ϫ1 Ϫ1 ever, heme concentration showed no change for a period of kii ഠ 0, and could be written as ഠ kiЈ . In this latter time but then a sharp decrease (pro®le II), whereas in the case, heme and CCl3R may participate in a type of interaction presence of 2,2,2-trichloroacetamide (R ϭ CONH2) and 2,2,2- other than electron transfer. It is possible that the nonbonding-
trichloroethanol (R ϭ CH2OH), heme concentration showed pair electrons on the oxygen [20] and/or nitrogen atoms [21] little or no variation (pro®le III). In these three cases, heme of the substituent can preferentially complex with the Fe(II) loss was attributed to the complexation and later oxidation site in competition with their chlorine atoms or participate in (except in pro®le III) of the iron(II) center to form chloro- pi-lone pair bonds with the ethylene groups located at the side Ϫ1 hemin. In all the experiments, trihalomethane was present in chains (kiii) so that ഠ kiЈϩk. iii molar excess regarding heme, or [CCl3R]0 k [heme]0, and heme oxidation for pro®les I and II was approximated as a Mechanism pseudo±®rst order reaction regarding heme. A detailed mechanism to explain these results is depicted A kinetic mechanism consistent with the results depicted in Figure 4. The ®rst step is the complexation of the CCl3R in Figure 3 would invoke two consecutive steps: fast com- molecule to the heme axial site through a chlorine atom. Chlo- plexation of CCl R with heme, followed by heme oxidation 3 rine atom charge transfer to the iron(II) center via overlap of via inner-sphere electron transfer (Eqn. 8). Applying the steady- an empty p and d8 orbital, respectively, will form a Fe-Cl state principle to formation of the precursor complex [17], the covalent bond. As [Fe(II)-Cl-CCl2R] forms, the Fe(II) transfers heme rate law expression can be rewritten as Equation 9: an electron to the bound chlorine to form Fe(III)Cl (chloro- ←→ki kii→ • • heme ϩ CCl33 R heme Ϫ CCl R P (8) hemin) and CCl2R. The radical CCl2R could then complex kϪi with another heme molecule to form Fe(II)±´CCl2R and un- Ϫd[heme] d[heme ´ ´ CCl R] dergo a second inner-sphere electron transfer to form ϭ 3 dt dt Fe(III)ϪϪ:CCl2R [6,7]. The participation of the chlorine as a bridging ligand supports the inner-sphere redox mechanism ϭ ki[heme][CCl3 R] [22]. At ®rst glance, Figure 4 suggests that the CCl R molecule Ϫ (k ϩ k )[heme ´ ´ CCl R] (9) 3 Ϫiii 3 undergoes electron transfers involving two heme molecules
where heme ´ ´ CCl3R refers to the precursor complex that and requires a minimum heme:CCl3R ratio of 2:1 for full con-
results from the binding of CCl3R to heme, ki to the adsorption version. Pentachloroethane (R ϭ CHCl2), however, undergoes rate constant of CCl3R, kϪi to the precursor complex dissoci- dechlorination in the presence of excess dithionite and a de- 546 Environ. Toxicol. Chem. 19, 2000 R.A. Larson and J. Cervini-Silva
Ϫ1 Fig. 5. Heme oxidation rate, kii (min ), as a function of R electro- negativity, R. Circles (pro®le I) refer to the trend observed when R Ϫ ϭ NO2, CHCl2, Cl, COO ,CH3, and H. Square (pro®le II) refer to the trend when R ϭ CN.
c. The carbon center acquires a different partial charge ac- cording to R's group electronic properties: A strongly electro- Fig. 4. Mechanism of reaction between heme and trichloromethanes in the presence of dithionite. negative group induces a charge de®ciency in the carbon atom that translates to a large (Fe Ϫc) value, and the electron ¯ow along the Fe-Cl-C bond shifts until (Fe Ϫc) approaches zero ®ciency of heme (CCl3CHCl2:dithionite:heme, 100:260:3) with (i.e., the ``electronegativity equalization'' [25±28]). Equaliza- a yield of as much as approximately 75% ([23]; J. Cervini- tion in electronegativity is followed by rupture of the Cl-C Silva et al., unpublished data). These results suggest that in- • bond for the formation of chlorohemin and a CCl2R radical. stead of acting as stoichiometric reagent, heme serves as an Presumably, the relative formation rate of the Fe-Cl bond is electron-transfer mediator. In this scenario, sulfoxyl radicals a function of (Fe Ϫc), and the rate of formation of chloro- act as reducing agents of Fe(III) and, perhaps, of Fe(III)Cl to • hemin and CCl2R radical depends on the electronic contri- form Fe(II). bution of R to stabilize the radical. For instance, acceptor Blank experiments in which dithionite alone was incubated Ϫ groups (NO2, CN, COO ) are substituents that possess high with pentachloroethane further showed that no dechlorination electronegativity to promote formation of the Fe-Cl bond, but products were formed, only polar products and, perhaps, sul- • they can also provide a higher CCl2R radical stability via odd- ®nic acids of the type reported elsewhere [24]. These exper- electron delocalization and form a three-electron bond [29]. iments demonstrated the necessity for heme to be present for On the other hand, acceptor groups (Cl, CH3, H) have a reductive dechlorination to occur. Fe c • moderate ( Ϫ) value and cannot contribute to CCl2R The stereochemistry of the bulk reductant may affect the radical stability, thus experiencing a slower dechlorination than mechanism of electron transfer between iron porphyrins and the acceptor groups. halogenated alkanes. For instance, dithionite is an ef®cient Figure 5 shows that the heme oxidation rate follows a strong electron source for iron [13], which in turn serves as an elec- relationship with R electronegativity, or R. In agreement with tron-transfer mediator in the presence of halogenated alkanes the results of previous studies [29±31], the electronegativity [23]. In the presence of dithionite, the formation of chloro- of a substituent appears to be an important variable to predict hemin supports the electron transfer between CCl3R and heme a trend in the reactivity of homologous compounds in a re- occurring via an inner-sphere mechanism, in agreement with action involving radical species (Table 1). The good correlation the results of previous studies in which Fe(0) was used as a R 2 of ln kii and (r ϭ 0.88; Eqn. 14) suggests no signi®cant bulk reductant [6,7]. Perlinger et al. [10], however, studied the deviation from this trend in reactivity that might cause the dehalogenation of polyhalomethanes and ethanes in the pres- conjugation of the R and chlorine electronic contributions (i.e., ence of iron(II) porphyrins and cysteine. They suggested an the ``captodative effect'' [32]). Heme oxidation in the presence outer- rather than an inner-sphere electron transfer (Eqns. 3 of trichloromethanes that follow pro®les I and II may be char- and 4) and hypothesized that cysteine blocks the axial site of the iron center. These studies suggest that bulk reductants Ϫ Table 1. Heme oxidation of CCl R, (minϪ1), and R (SO2• vs cysteine) may compete for the electron-transfer site 3 kii R with trichloromethanes and modify the mechanism of reaction. electronegativity,
This may have signi®cant implications in pathways of cellular Ϫ1 Ra Compounds R kii (min ) detoxi®cation and iron transport as well as in bioremediation of water bodies and soils. CCl3NO2 NO2 Ͼ3.71 3.45 CCl3CHCl2 CHCl2 0.39 2.88 R electronegativity and carbon-centered radical stability CCl3CN Cn 0.4570 3.17 CCl4 Cl 0.09 3.03 Ϫ Ϫ The inner-sphere electron transfer between heme and CCl3R CCl3COO COO 0.06 2.92 (kii) includes the electron transfer and the formation of chlo- CCl3CH3 CH3 0.03 2.63 rohemin and •CCl R radical. As CCl R and heme molecules CHCl3 H 0.012 2.28 2 3 CCl C(O)NH C(O)NH Not observed 2.84 approach each other and complex, there is charge transfer 3 2 2 CCl CH OH CH OH Not observed 1.78 along the Fe-Cl-C bond that is driven by the difference in the 3 2 2 electronegativity of the iron and the carbon atoms, or Fe Ϫ a Data from Wells [30] and Willmshurst [31]. Dechlorination of substituted trichloromethanes Environ. Toxicol. Chem. 19, 2000 547 acterized by the same function (Fig. 5), and this supports the port of fellowships from the National Council of Science and Tech- hypothesis that pro®le II is a particular case of pro®le I. These nology, Mexico, CONACyT-92807, and the National University of Mexico, DGAPA-UNAM. R.A. Larson and J. Cervini-Silva thank results con®rm that the electronegativity of a substituent is a Karen Marley for helpful discussions and technical assistance. helpful property in predicting the reduction rate of a homol- ogous series of substrates by a metal center [28±30]: REFERENCES
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