Monofluorinated Analogues of Polybrominated Diphenyl Ethers as

CHIRON AS, Stiklestadveien 1, N-7041 Trondheim, Norway, and Department of Occupational and Environmental Health,. University of Iowa, Iowa City, Iowa ...
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Environ. Sci. Technol. 2006, 40, 3023-3029

Monofluorinated Analogues of Polybrominated Diphenyl Ethers as Analytical Standards: Synthesis, NMR, and GC-MS Characterization and Molecular Orbital Studies G R E G O R L U T H E , * ,‡ P I M E . G . L E O N A R D S , §,† GABY S. REIJERINK,⊥ HULING LIU,⊥ JON E. JOHANSEN,⊥ AND LARRY W. ROBERTSON| Institute of Chemistry, Norwegian University of Science and Technology, N-7041 Trondheim, Norway, RIVO, Netherlands Institute for Fisheries Research, IJmuiden, The Netherlands, CHIRON AS, Stiklestadveien 1, N-7041 Trondheim, Norway, and Department of Occupational and Environmental Health, University of Iowa, Iowa City, Iowa 52242

Polybrominated diphenyl ethers (PBDEs), a group of 209 individual congeners distinguishable by the number and position of bromines, are produced for use as flame retardants in consumer goods. PBDEs have become ubiquitous environmental contaminants, present in increasing levels in the environment and humans. In the present study, 10 individual monofluorinated analogues of PBDEs (FPBDEs) and one difluorinated PBDE (FF-PBDE) were synthesized and characterized, and their gas chromatographic (GC) and mass spectrometric (MS) characteristics determined. The synthesis method utilized a nucleophilic reaction of bromophenols with diphenyliodonium salts and the perbromination of fluorosubstituted diphenyl ethers. Reaction yields were between 10% and 59% with g98% purity. Apart from the aromatic ring carrying the fluorine atom, only minor chemical nuclear magnetic resonance (NMR) shift changes were observed in comparison to the corresponding parent PBDEs, with the exception that the JF,H coupling was stronger. Our preliminary data show that F-PBDEs and PBDEs have comparable retention times in gas chromatography with F-PBDEs demonstrating in general shorter or identical retention times, depending on the pattern of fluorine substitution. We also calculated the torsion angles and the dipole moments for both and report that there is a good correlation between GC retention times and the torsion angles but not with dipole moments. In MS, the difference of the ion peaks of the F-PBDE/ PBDE pairs is m/z 19 (F), which allows a simultaneous MS detection without separation. On the basis of GC * Corresponding author current address: Department of Occupational and Environmental Health, University of Iowa, 100 Oakdale Campus, # 124 IREH, Iowa City, IA 52242-5000; phone: +1 319 335 4221; fax: +1 319 335 4290; e-mail: [email protected]. ‡ Norwegian University of Science and Technology. † Current address: Institute for Environmental Studies (IVM), Free University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. § Netherlands Institute for Fisheries Research. ⊥ CHIRON AS. | University of Iowa. 10.1021/es052410z CCC: $33.50 Published on Web 03/29/2006

 2006 American Chemical Society

separation, simultaneous MS detection, and the stability of fluorine due to its generally resistance to nucleophilic displacement, we propose that F-PBDEs may function as valuable potential standards, markers, and tracers in environmental analysis.

Introduction Polybrominated diphenyl ethers (PBDEs) are a group of 209 individual congeners, distinguishable by the number and position of bromines in the rings. PBDEs have been industrially produced and widely used over the past 2 decades as additive flame retardants in consumer goods, for example, high-impact polystyrene, foams, textiles, wire and cable insulation, and conductors (1, 2). Despite their strong flameretardant properties, PBDEs seem to be migrating from the products in which they are used and are accumulating in the environment (3, 4). PBDEs are now ubiquitous contaminants, and due to their lipophilic character, high bioavailability, and slow elimination, they bioaccumulate in the body and biomagnify in the food web (5-12). As a result of their widespread use and their entry into the environment, PBDEs can be found in higher organisms, including humans (6-13). The environmental levels of PBDEs are in general still lower than those of polychlorinated biphenyls (PCBs). However, while the levels of PCBs and many other chlorinated contaminants are decreasing, the levels of PBDEs are increasing. PBDE levels found in people living in North America are currently 10-100 times higher than those in Europe or Japan and are doubling every 4-6 years (7, 11). Time trends have further shown that the Swedish responses to PBDEs were effective: PBDE body burdens began to trend downward following the phase out of the penta-PBDE flame retardant in the late 1990s (14). Gas chromatography (GC) coupled mass spectromety (MS) in electron capture negative chemical ionization (ECNI) mode and electron ionization (EI) are the approaches most frequently used for PBDE analyses (15, 16). GC retention behavior is dependent on the physical properties of PBDEs, including conformation. The conformations of diphenyl ethers are described by the torsional angles (φ1 and φ2) between the C1-O-C1′ plane and planes of the phenyl rings. The angles are defined as positive when the rotation is clockwise looking down the C4-C1 and C4′-C1′ axes toward the oxygen. Conformational properties of variously substituted diphenyl ethers have previously been studied using semiempirical or ab initio calculations (17). To our knowledge, however, all the studies with regard to diphenyl ethers have essentially not covered PBDEs except several recent investigations on PBDEs 51 and 166 (18). Theoretically, there are four possible types of conformations of diphenyl ethers: planar (φ1 ) φ2 ) 0°), butterfly (φ1 ) φ2 ) 90°), skew (φ1) 0°, φ2) 90°), and twist (φ1, φ2 > 0°). A variety of experimental and theoretical studies have shown that diphenyl ether has a twist conformation in that φ1 and φ2 lie in the vicinity of 25-50° (19). It has been shown that the PBDE congeners prefer a skew or twist conformation depending on the number of the ortho substituents (17). Conformational properties of PBDEs are of special importancesaside from their mechanisms in molecular recognition in the macromolecular bindingsto understand boiling points and by this retention behavior in GC. In addition the dipole moments are a factor that might have a stronger influence. MS strongly depends on the type of ionization used. ECNIMS is by far the most popular technique because it provides VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Synthesis of the diphenyliodonium salts (3a-c) from bromobenzenes (1a-c) and the synthesis of F-PBDEs (5a-i) by nucleophilic aromatic substitution of the diphenyliodonium salts (3a-c) with bromophenols (4a-g). high levels of sensitivity. NCI mass spectra of all PBDEs are dominated by the bromine ion [Br]-, and the molecular ion is insignificant. By contrast, EI provides better structural information, giving the molecular ions and the sequential losses of bromine atoms. In ECNI-MS experiments, the two ions corresponding to m/z ) 79 and 81 [Br]- are monitored, whereas, in EI-MS experiments, the two most abundant isotope peaks for each level of bromination correspond to molecular clusters. Moreover, in the EI-MS approach and because of the availability of 13C-labeled standards, methods were developed based on the quantification by the isotopic dilution technique. The main advantage of ECNI-MS versus EI-MS is the limits of detection (LODs) afforded, but higher specificity and accuracy can be obtained using EI-MS. However, both ionization modes are subjected to different types of interferences. In general, EI-MS is affected by chlorinated interferences, especially polychlorinated biphenyls (PCBs). ECNI-MS eliminates chlorinated interferences, but different brominated interferences are well-resolved with EI-MS. Based on animal studies, PBDEs may cause liver toxicity, disruption of thyroid hormone levels (20, 21), neurotoxicity (22, 23), and reproductive and developmental toxicity (2225). In addition PBDE metabolites may interact with cellular macromolecules, such as DNA (26), and could thereby be carcinogenic. There is an increasing interest in the trace analysis of PBDEs, sample preparation and final determination of which may well lead to systematic and nonsystematic (random) errors. It is generally accepted that the accurate determination of micro contaminants in such complex mixtures requires the use of external or, preferably, internal standards (ISs). The benefit of using ISs with physicochemical properties similar to those of the target compounds is that both types of errors will be minimized. Aside from the assistance in analytical processes, tracers and markers are needed in mechanistic, environmental, and metabolic studies to investigate pathways. Since earlier work on monofluorinated analogues of polycyclic aromatic hydrocarbons (F-PAHs) was very promising (27, 28), we have now synthesized a series of monofluorinated analogues of PBDEs (F-PBDEs) and one difluorinated PBDE (FF-PBDE). Here we present their synthesis and characterization by means of 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and their chromatographic (GC) and mass spectrometric (MS) characteristics in electon ionization (EI) and electron capture negative chemical ionization (ECNI) mode. In additon, we calculated the conformations and the dipole moments of the F-PBDEs and the FF-PBDE and compared them with the corresponding 3024

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parent PBDEs. Altogether three procedures have been used for the synthesis of individual F-PBDEs: (i) coupling of a fluorophenol with a bromobenzene; (ii) bromination of fluorodiphenyl ether; (iii) coupling of a fluorinated or brominated diphenyliodonium salt with a fluoro- or bromophenol. We address the hypothesis that F-PBDEs resemble their corresponding nonfluorinated parent PBDEs very closely in physicochemical properties. For this purpose, we present the GC-ECNI-MS of PBDE and F-PBDEs. F-PBDEs show advantages when using ECNI-MS detection because the dominant ions for F-PBDEs, 13C-PBDEs and PBDEs are m/z 79 and 81, and a separation by retention time between the internal standards (F-PBDEs or 13C-PBDEs) and PBDEs is, therefore, necessary.

Experimental Section Chemicals. The substituted bromobenzenes (1a-c), bromophenols (4a-h), bromine (p.a.), and aluminum bromide were purchased from Acros (Geel, Belgium). Iodine (p.a.), sulfuric acid (p.a.), fuming sulfuric acid (p.a.), hydrochloric acid (p.a.), fumic nitric acid (p.a.), sodium hydroxide (p.a.), methanol (p.a.), and silica gel 60 Å C:C 40-63 µm were purchased from J. T. Baker (Deventer, The Netherlands); n-hexane was purchased from Riedel de Hae¨n (Seelze, Germany); CDCl3, DMSO-d6, and TMS were purchased from Sigma-Aldrich (St. Louis, MO). Synthesis. General Procedure for the Formation of Diphenyliodonium Chlorides. According to the procedure described previously (29), iodine (1.59 g, 6.25 mmol) was added to sulfuric acid (1.9 mL) in the presence of fuming sulfuric acid (30%, 3.75 mL), and fumic nitric acid (100%, 0.80 mL) was slowly added. The reaction mixture was stirred at 70-80 °C for 2 h, at which time yellow crystals of iodyl sulfate precipitated. The mixture was than cooled to 0 °C. Under vigorous stirring the bromobenzenes (1a-c) (32.3 mmol) were slowly added to the iodyl sulfate (IO)2SO4. The mixture was stirred at 45 °C for 2 h and then cooled to 0 °C. Ice (10 g) was added carefully in small portions [Caution: exothermic reaction!]. The nitrogen oxides were removed by introducing a gentle steam of nitrogen through the reaction solution. The product, a brown oil, was collected, dissolved in methanol, and crystallized as the chloride salt by dropwise addition of concentrated hydrochloric acid After the reaction was completed, the reaction mixture was maintained for an additional 2 h to give the diphenyliodonium chlorides (3ac). See Figure 1 for the general equation. (3a) Yield 63%; 98% purity; 1H NMR (DMSO-d6) δ 8.67 (2H, dd, J ) 2.1 and 6.5 Hz), 8.26 (2H, m), and 7.52 (2H, t, J ) 8.8 Hz). 13C NMR

TABLE 1. Synthesis of Single F-PBDE Congeners (5a-j) by Coupling of Diphenyliodonium Salts (3a-c) with Bromophenols (4a-g), Synthesis of Fluorinated Diphenyl Ethers 0-4′,6F (6a) and 0-4′F (6b), and the Bromination To Obtain 201-4′,6F (5j) and 208-4′F (5k) yield no.

F-PBDE BZ-no.

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 6a 6b

25-4′F 27-4′F 28-3′F 47-6F 66-6F 69-4′F 100-3F 119-3F 160-4′F 201-4′,6F 208-4′F 0-4′,6F 0-4′F

substitution pattern

diphenyliodonium salt

2,3′,4-triBr-4′F 2,3′,6-triBr-4′F 2,4,4′-triBr-3′F 2,2′,4,4′tetraBr-6F 2,3′,4,4′-tetraBr-6F 2,3′,4,6-tetraBr-4′F 2,2′,4,4′,6-pentaBr-3F 2,3′,4,4′,6-pentaBr-3F 2,3,3′,4,5,6-hexaBr-4′F 2,2′,3,3′,4,5,5′,6′-octaBr-4′,6F 2,2′,3,3′,4,5,5′,6,6′-nonaBr-4′F 4′,6F 4′F

3a 3a 3b 3b 3c 3a 3b 3c 3a bromination of 6a bromination of 6b coupling of 4h with 1a coupling of 4h with 1b

bromophenols 4a 4b 4f 4e 4e 4c 4g 4g 4d

purified [%]

purity [%]

59 47 53 57 50 10 52 55 10 29 20 48 52

99 98 98 98 99 98 98 99 99 98 98

TABLE 2. 1H Chemical Shifts (δ [ppm] Relative to TMS) and 3J(H,H), 3J(F,H), 4J(H,H), and 4J(F,H) Couplings, Mass Spectral Data Using ECNI as Ionization Mode of F-PBDE Congeners 25-4′F (5a), 27-4′F (5b), 28-3′F (5c), 47-6F (5d), 66-6F (5e), 69-4′F (5f), 100-3F (5g), 119-3F (5h), and 160-4′F (5i), and Relative Abundance to m/z 79 of m/z Values That Are Higher than 3% of m/z 79 1H

F-PBDE

MS characterization (relative abundance [%] of m/z values to m/z 79)

NMR characterization, shifts δ [ppm], coupling constants J [Hz]

no.

BZ-no.

5a

25-4′F

5b

27-4′F

5c

28-3′F

5d

47-6F

5e

66-6F

5f

69-4′F

5g

100-3F

5h

119-3F

δ 7.87 (1H, d, J ) 6.9 Hz), 7.53 (1H, d, J ) 8.7 Hz), 7.09 (1H, d, J ) 2.9 Hz), 6.65 (1H, dd, J ) 2.9 and 8.8 Hz)

5i

160-4′F

δ 7.00 (1H, dd, J ) 7.9 and 9.0 Hz), 6.93 (1H, dd, J ) 3.1 and 5.5 Hz), 6.63 (1H, ddd, J ) 3.1, 3.6 and 9.0 Hz)

δ 7.79 (1H, d, J ) 2.3 Hz), 7.42 (1H, dd, J ) 2.3 and 8.7), 7.25 (1H, dd, J ) 3.0 and 5.5 Hz), 7.11 (1H, dd, J ) 7.9 and 9.0 Hz), 6.90 (1H, m) and 6.84 (1H, d, J ) 8.7) δ 7.63 (1H, d, J ) 2.4 Hz), 7.38 (1H, t, J ) 2.7 Hz), 7.35 (1H, t, J ) 2.0 Hz), 7.23 (1H, dt, J ) 2.0, 6.7 Hz), 6.84 (1H, t, J ) 8.7 Hz), 6.72 (1H, d, J ) 8.7 Hz). δ 7.78 (2H, d, J ) 8.1 Hz), 7.06 (1H, t, J ) 8.0 Hz), 7.05 (1H, dd, J ) 8.1 and 9.3 Hz), 7.00 (1H, dd, J ) 2.5, 5.6 Hz), 6.74 (1H, m) δ 7.77 (1H, d, J ) 2.3 Hz), 7.62 (1H, dd, J ) 2.1 and 4.1), 7.36 (1H, dd, J ) 2.3 and 9.0 Hz), 7.27 (1H, dd, J ) 2.4 and 10 Hz), 6.42 (1H, dd, J ) 1.1 and 8.0) δ 7.62 (1H, dd, J ) 2.0and 4.2 Hz), 7.52 (1H, d, J ) 8.8 Hz), 7.36 (1H, dd, J ) 2.2 and 9.0 Hz), 7.14 (1H, d, J ) 3.0 Hz) 6.72 (1H, dd, J ) 3.0 and 9.0) δ 7.78 (2H, s), 6.93 (1H, dd, J ) 7.9 and 8.9 Hz), 7.00 (1H, dd, J ) 3.0 and 5.4 Hz), 6.63 (1H, ddd, J ) 3.2, 3.6 and 9.0 Hz) δ 7.87 (1H, d, J ) 6.9 Hz), 7.80 (1H, d, J ) 2.4 Hz), 7.27 (1H, dd, J ) 2.4 and 8.8 Hz), 6.26 (1H, d, J ) 8.8 Hz)

(DMSO-d6) δ 160.0 (C-4, C-4′, d, 1JC-F ) 249.0 Hz), 130.5 (C-1, C-1′, s), 136.6 (C-2, C-2′, d, 3JC-F ) 8.25 Hz), 119.8 (C-5, C-5′, d, 2JC-F ) 23.3 Hz), 116.4 (C-6, C-6′, s), 110.4 (C-3, C-3′, d, 2J 1 C-F ) 21.8 Hz). (3b) Yield 54%; 98% purity; H NMR (DMSOd6) δ 8.32 (2H, d, J ) 8.0 Hz), 8.25 (2H, d, J ) 2.4 Hz), 7.68 (2H, dd, J ) 2.0 and 8.0 Hz). (3c) Yield 60%; 96% purity; 1H NMR(DMSO-d6) δ 8.65 (2H, d, J ) 2.0 Hz), 8.10 (2H, dd, J ) 2.0 and 8.4 Hz), 7.86 (1H, d, J ) 8.0 Hz). General Procedure for the Nucleophilic Substitution of Diphenyliodonium Chlorides by Bromophenols. The diphenyliodonium chlorides were produced as previously described (29). The obtained diphenyliodonium chlorides (3a-c) (2.5 mmol) were coupled with the bromophenols (2.5 mmol) in the presence of an aqueous solution (20 mL) of sodium

161 (42), 159 (21), 81 (95), 79 (100) 161 (60), 159 (30), 81 (95), 79 (100) 345 (3), 343 (10), 341 (3), 161 (42), 159 (21), 81 (95), 79 (100) 345 (9), 343 (18), 341 (9), 161 (42), 159 (25), 81 (95), 79 (100) 345 (2), 343 (4), 341 (2), 161 (42), 159 (23), 81 (95), 79 (100) 424 (8), 423 (25), 421 (27), 419 (8), 345 (6), 343 (8), 341 (6), 161 (19), 159 (9), 81 (95), 79 (100) 424 (3), 423 (7), 421 (8), 419 (3), 345 (6), 343 (8), 341 (6), 161 (19), 159 (9), 81 (95), 79 (100) 583 (3), 581 (8), 579 (2), 504 (5), 502 (8), 500 (4), 424 (3), 423 (4), 421 (4), 419 (3), 345 (2), 343 (3), 341 (2), 161 (13), 159 (7), 81 (95), 79 (100) 490 (72), 488 (120), 486 (140), 484 (72), 429 (23), 427 (41), 425 (23), 412 (30), 410 (50), 408 (63), 406 (27), 161 (5), 159 (3), 81 (95), 79 (100)

hydroxide (0.1 g, 2.5 mmol), and the mixture was refluxed. The reaction was monitored by means of GC-FID and TLC. The reaction mixture was maintained under stirring for 5 h before being extracted with diethyl ether (3 × 50 mL). The organic layers were combined, washed with water, dried with MgSO4, and filtered and the solvent was evaporated. The crude product was purified by flash chromatography over silica gel with n-hexane as mobile phase. The purified F-PBDEs (5a-k) were dried with a high vacuum oil pump. See Figure 1 for the general procedure and Table 1 for the reaction yields. See Table 2 for the 1H NMR and MS characterization of the F-PBDEs (5a-k), and Table 3, Supporting Information (SI), for the 13C NMR (5a,d,j,k). VOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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General Procedure for the Nucleophilic Substitution of Bromobenzene and 2-Fluorobromobenzene by 4-Fluorophenol. A mixture of 4-fluorophenol (4h) (10 mmol), potassium hydroxide (0.56 g, 10 mmol), 2-fluorobromobenzene (1a) or bromobenzene (1b) (12 mmol), and copper powder (0.64 g, 10 mmol) was heated at 110 °C for 2 h. The residual bromobenzenes were removed by distillation under reduced pressure, and the obtained diphenyl ethers (6a,b) were purified on a silica gel column with n-hexane as mobile phase. See Table 1 for the yields. General Procedure for the Perbromination of Fluorinated Dibenzoethers. The reaction was performed according to literature descriptions (30-33). Bromine (1.4 mL, 27.2 mmol) was slowly added to the diphenyl ethers (6a,b) (2.43 mmol) and aluminum bromide (0.04 g, 0.15 mmol). After the addition was completed, the mixture was heated to 59 °C for 12 h. The reaction was stopped by addition of an aqueous solution of NaHSO3. The organic phase was separated, washed with water, and dried over sodium sulfate. The crude product was purified by recrystallization from methanol. See Table 1 for the yields. Chemical Analysis. Synthesis Monitoring and Purification Procedure. The reactions were monitored and the purity of all compounds was determined by GC and thin-layer chromatography (TLC). A GC (Varian 3800 Palo Alto, CA) equipped with a flame ionization detector (FID) was used. Separation was performed on a CP-sil 5CB column (Chrompack, Darmstadt, Germany), 20 m × 0.25 mm i.d., 0.25 µm film thickness; nitrogen was used as carrier gas. The column temperature was programmed from 50 °C (hold 2 min) to 300 °C (20 min) at 15 °C/min. For TLC, ALUGRAM SIL G/UV254 (Machery-Nagel, Du ¨ ren, Germany) was used as stationary and n-hexane as mobile phase. Flash chromatography was the method of choice for the purification processes. An open column was used as well as a medium-pressure liquid chromatograph (MPLC) (Baeckstro¨m SEPARO AB, Lidingo¨, Sweden). Both systems use silica gel as stationary and n-hexane as mobile phase. For 1 g of crude product, ca. 300 g of silica gel was used. 1H and 13C NMR and MS Characterization. All F-PBDE congeners were characterized by means of 1H or 13C NMR or both and MS. The 1H and 13C NMR spectra were recorded on 400 MHz NMR spectrometer (Bruker, Billerica, MA), using CDCl3 and DMSO-d6 as solvents. Chemical shifts, δ, are given in ppm relative to TMS; coupling constants, J, are given in Hz. Analysis of F-PBDEs and PBDEs were carried out on a HP 6890 (Agilent, Palo Alto, CA) GC with MS detection and autosampler (Agilent). Briefly, 1 µL was injected splitless, 1 ug absolute. The injection temperature was set to 275 °C. Separation was performed on a CP Sil 8 capillary column (50 m × 0.25 mm i.d., 0.25 µm film thickness), except for the nona-BDEs 208 and 208-4′F where a DB-5 (15 m × 0.25 mm i.d., 0.25 µm film thickness) was used. Helium was used as the carrier gas at a flow of 1.2 mL/min. The split was opened after 2 min. The column temperature for the CP Sil-8 was programmed from 90 °C (3 min) to 210 °C at 30 °C/min (20 min) followed to 290 °C (15 min). The final temperature was held for 15 min. For DB-5, the column temperature was 90 °C (3 min) to 300 °C at 30 °C/min (20 min). Detection was based on ECNI-MS in the full scan mode (m/z 70-850). Methane was used as reagent gas at a flow of 3 mL/min. The ion source temperature was 200 °C. Molecular Orbital Calculations. To calculate the conformation and the dipole moments of the F-PBDEs and their corresponding parent PBDEs, semiempirical SCF-MO calculations using an AM1 Hamiltonian (34) with the Spartan ’02 package were carried out on Quad 2.5 GHz Power Mac G5 with a PCI express graphic card. The heats of formation were computed using a starting geometry similar to an optimized geometry. The use of symmetry constraints 3026

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enhanced the convergence compared to completely unconstrained runs. The conformations of diphenyl ethers are described by the torsional angles (φ1 and φ2) between the C1-O-C1′ plane and planes of the phenyl rings. The angles are defined as positive when the rotation is clockwise looking down the C4-C1 and C4′-C1′ axes toward the oxygen. The changes in the conformation by the introduction of a fluorine were calculated. The semiempirical SCF-MO calculations using an AM1 were applied as well to calculate dipole moments of the F-PBDEs and PBDEs. Changes in the dipole moment were calculated by subtraction of the values for F-PBDEs from the PBDEs. So a positive value indicates that the F-PBDEs have a smaller overall dipole moment than the corresponding PBDE. Deviation values obtained by two other calculation methods are around 5-10%. Table 4 gives an overview of the torsion angles, differences, bond angles, dipole moments, and differences.

Results and Discussion Synthesis Aspects. In this paper, we report the synthesis of 10 F-PBDEs and one difluorinated PBDE (FF-PBDE), 2014′6F (5j). These F-PBDEs and FF-PBDE cover the range of substitution from three up to nine bromine atoms. F-PBDEs can be prepared by using different methods. With the exceptions of the F-PBDEs 201-4′,6F (5j) and 208-4′F (5k), all F-PBDEs (5a-i) were synthesized by a nucleophilic aromatic substitution of diphenyliodonium salts (3a-c) by phenols (4a-g). The diphenyliodonium salts (3a-c) themselves were synthesized according to Marsh et. al (29) by an electrophilic aromatic substitution of (fluoro)bromobenzenes (1a-c) with iodyl sulfate (IO)2SO4 as the electrophile. The yields obtained are comparable to those found in the literature: 2,2′,4,4′-tetrabromodiphenyliodonium chloride (3b) 54-62% and 3,3′,4,4′-tetrabromodiphenyliodonium chloride (3c) 60-57%. F-PBDEs 25-4′F (5a), 27-4′F (5b), 694′F (5f), and 160-4′F (5i) are fluorosubstituted in the para (4 and 4′) positions. This is due to the regioselectivity of fluorine in the electrophilic aromatic substitution during the formation of the iodonium salt (3a). Fluorine is ortho- and paratruing because of the -I and +M effect. The -I effect decreases strongly with distance, so the para-truing becomes dominant. The +M effect stabilizes the reactive arenium ion intermediate by resonance. Comparing the yields of F-PBDEs that we obtained with literature (29) yields for PBDEs, we find similar results and in specific cases higher ones. This is true for F-PBDEs that are fluorine substitued in the 3 and 3′ positions: 28-3′F (5c) (53%) compared with 28 (16%), 100-3F (5g) with 100, and 119-3F (55%) with 119 (8%). This finding cannot be explained by the basicity of the phenols because the pKa of 3-fluorophenol (9.21) is lower than of phenol (10). But the nucleophilicity depends as well on the solvation, especially in a protic solvent. Additionally, fluorine increases the electron density in the ortho and para positions of the resonance structures of the phenolates. These might be the reasons for the higher yields for the monofluorinated analogues. In general we found the nucleophilic aromatic substitution of a (fluoro-)bromo-diphenyliodonium chloride with a (fluoro-)bromophenol is the method of choice to synthesize milligram quantities of F-PBDEs. These reactions result in F-PBDEs with high purity and determined impurities (other congeners). This is specifically important for biological and toxicological studies. Higher brominated congeners 201-4′,6F (5j) and 208-4′F (5k) were synthesized by perbromination of fluorinated diphenyl ethers (6a,b) according to the methods used by Golounin et al. (32). In combination with the application of protecting groups, for example, amino groups (33), this procedure will be the method of choice to obtain further higher brominated congeners. The amine can be diazotized

TABLE 4. Computed Values for F-PBDEs and PBDEs of Their Torsion Angles, Their Difference, and Their Bond Angles [deg] and Their Dipole Moments [debye] and Differences [∆]a F-PBDE and PBDE no. 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k a

dipole moment and differences

torsion angle and differences

BZ-no.

φ1 C1′-O-C1-C6

25 25-4′F 27 27-4′F 28 28-3′F 47 47-6F 66 66-6F 69 69-4′F 100 100-3F 119 119-3F 160 160-4′F 201 201-4′6F 208 208-4′F

42.8 45.1 93.5 93.5 43.5 54.3 -42.2 72.5 30.0 -110.9 (93.5 (93.5 106.6/-80.5 107.6 (93.5 -94.4 (93.5 (93.5 -138.8 -133.8 54.9/-132.5 -53.9/133.4

|∆φ1| 2.3 0.0 10.8 30.3 80.9 0.0 1.0/27.1 0.9 0.0 5.0 1.0/1.1

φ2 C2′-C1′-O-C1 -148.4 -149.1 180.0 180.0 -151.4 -159.7 142.8 -155.3 137.1 -162.3 180.0 180.0 162.6 160.7 0.0 1.6 0.0 0.0 -127.5/60.3 128.2 / -59.0 56.0/-131.6 -57.6/130.2

|∆φ2| 0.7 0.0 8.3 12.5 25.2 0.0 1.9 1.6 0.0 0.7/1.3 1.6/1.4

bond angle C1-O-C1′

δ

116.5 116.3 116.4 116.3 116.7 116.6 116.1 116.3 116.5 116.4 116.4 116.4 116.4 116.4 116.6 116.6 116.5 116.5 119.0 118.2 119.0 119.0

2.23 1.70 3.54 4.17 1.20 2.33 1.98 2.37 1.91 2.72 2.29 2.73 2.22 1.95 1.39 1.37 0.89 1.04 1.26 0.88 1.25 0.35

∆δ (PBDE - F-PBDE) 0.53 -0.63 -1.13 -0.39 -0.81 -0.44 0.27 0.02 -0.15 0.38 0.9

For specific data, see Experimental Section.

and transformed into various functional groups or just reduced using Sandmeyer reactions. All F-PBDEs (5a-k) could be obtained with a purity g98% (n ) 5) according to GC analysis, with the exception of 201-4′,6F (5j) and 208-4′F (5k). The direct bromination of fluorinated diphenyl ethers can lead to congeners with variable grades of bromination with various substitution patterns. This disadvantage makes the separation and purification of the desired product difficult, if not impossible. To synthesize the target F-PBDEs congeners, there is a strong need of further investigation of specific methods. On the basis of the synthesis procedures, but also for the desired studies of the behavior of F-PBDEs, we synthesized congeners with fluorosubstitution in ortho (2, 2′, 6, 6′), meta (3, 3′, 5, 5′), and para positions (4, 4′). F-PBDEs. The following F-PBDEs were synthesized: 4′fluoro-2,3′,4-tribromodiphenyl ether, PBDE 25-4′F (5a); 4′fluoro-2,3′,6-tribromodiphenyl ether, PBDE 27-4′F (5b); 3′fluoro-2,4,4′-tribromodiphenyl ether, PBDE 28-3′F (5c); 6-fluoro-2,2′,4,4′-tetrabromodiphenyl ether, PBDE 47-6F (5d); 6-fluoro-2,3′,4,4′-tetrabromodiphenyl ether, PBDE 66-6F (5e); 4′-fluoro-2,3′,4,6-tetrabromodiphenyl ether, PBDE 69-4′F (5f); 3-fluoro-2,2′,4,4′,6-pentabromodiphenyl ether, PBDE 1003F (5g); 3-fluoro-2,3′,4,4′,6-pentabromodiphenyl ether, PBDE 119-3F (5h); 4′-fluoro-2,3,3′,4,5,6-hexabromodiphenyl ether, PBDE 160-4′F (5i); 4′,6-difluoro-2,2′,3,3′,4,5,5′,6′-octabromodiphenyl ether, PBDE 201-4′,6F (5j); 4′-fluoro-2,2′,3,3′,4,5,5′,6,6′-nonabromodiphenyl ether, PBDE 208-4′F (5k). The numbering is according to a previously used nomenclature (35-37). For an overview, see Table 1 as well. 1H and 13C NMR Characterization. Characterization of the F-PBDEs (5a-i) was carried out by means of 1H and 13C NMR of 25-4′F (5a), 47-6F (5d), 201-4′,6F (5j), and 208-4′F (5k). The perhalogenated F-PBDEs could only be characterized by 13C NMR. The results are listed in Tables 3 and 4, showing the chemical shifts, δ, measured in ppm and their couplings, J, measured in Hz. The 1H δ shifts vary from 6.26 ppm up to 7.87 ppm. Apart from the aromatic ring carrying the fluorine atom, only minor chemical shift changes were

observed in comparison to the corresponding parent PBDEs, with the exception that the JF,H coupling is stronger, between 4J ) 2.3 Hz and 3J ) 9.0 Hz. For a detailed characterization and interpretation, the one-dimensional 1H NMR spectra are too complex. Therefore we will perform specific 1H-13C correlated and J resolved spectra. The changes in the electron distributions can be indicated from the 13C NMR measurements. The 13C NMR δ shifts vary from 109.7 to 158.8 ppm, see Table 3 (SI). The carbon atom with the fluorine substitution, ipso-C, shows the strongest low field shift. The carbons in ortho positions to the fluorine are strongest high field shifted. Physicochemical Properties of Fluorine-Tagged PBDEs, F-PBDEs. The physical and chemical properties of Fcontaining organic compounds are related to a number of distinctive characteristics of the fluorine atom and include its high electronegativity, its radius, which is much smaller than that of the homologue chlorine, the strength of the C-F bond, and the absence of oxidation levels higher than F0. The combination of these characteristics dictates that fluorinated organic compounds closely resemble hydrocarbons in molecular size, are highly resistant to the nucleophilic displacement of fluoride, and are rather stable. Nucleophilic aromatic substitution is an exception to this. Here fluorine functions as a good leaving group and can be displaced. For this the aromatic ring must carry several strong electronwithdrawing groups, for example, in 2,4-dinitrofluorobenzene (Sanger reagent). In F-PBDEs, fluorine cannot be displaced by this mechanism. The unusually small but desirable effect of fluorine monosubstitution on the properties of aromatic hydrocarbons, like F-PBDEs, can be qualitatively understood by considering the influence of a fluorine atom on an aromatic system. First, fluorine induces a dipole moment, though not as strong as one might expect. The small fluorine atom is too hard to retain its complete electron charge. By hyperconjugation, caused by overlap of the aromatic π-orbitals and the free fluorine p-orbitals, this results in a partial backtransfer of electron density (38). Second, regarding interVOL. 40, NO. 9, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Typical chromatogram (GC-EI-MS) of nine F-PBDE and native PBDE pairs (50 ng/mL each in iso-octane). Conditions: m/z 35-400. molecular interactions as a result of the reduced polarizability, the London forces are less strong for F-PBDEs than for the parent PBDEs. We calculated the conformation of the F-PBDEs and PBDEs with a semiempirical SCF-MO method using AM1 (see Table 4). The conformational analysis by Nevalainen and Rissanen (17), based on energy maps calculated by this method, demonstrated good agreement with the X-ray diffraction data, and this prompted our current AM1 investigation of PBDEs. According to the torsion angles, the screw conformation is the most stable one. The energy minima of F-PBDEs are highly dependent on the position of the fluorine; we found that para fluorine substitution changes the ∆φ1 and ∆φ2 between 0.0° and 2.3° for ∆ (25-4′F (5a), 25), meta between ∆φ1 and ∆φ2 0.9° (119-3F (5h), 119) and 10.8° and ortho between ∆φ1 and ∆φ2 12.5° (47-6F (5d), 47) and 80.9° (66-6F (5e), 66). Further the bond angle of all F-PBDEs and PBDEs is between 116.1° (47) and 116.7° (28), with the exception of the highly brominated F-PBDEs, for example, 118.2° (201-4′,6F (5j)) and 119.0° (208-4′F (5k)), and PBDEs, 119.0° (201 and 208). The bond is widened due to the strong steric hindrance in highly brominated PBDEs and F-PBDEs. The results of the calculations of the dipole moments show that F-PBDEs and their corresponding parent PBDEs can have similar values, for example, 119-3F and 119; F-PBDEs can have a higher dipole moment, for example, 27-4F (5b), 28-3F (5c), 47-6F (5d), 66-6F (5e), 69-4′F (5f), and 160-4′F (5i), or lower one, for example, 25-4′F (5a), 100-3F (5 g), 201-4′6F (5j), and 208-4′F (5k). The dipole moment was dependent on the total pattern of substitution, including bromine and fluorine, and the conformation. GC-NCI-MS and GC-EI-MS Characteristics. As shown in Figure 2, all F-PBDEs in general have shorter or identical retention times because they are slightly less polar. The elution order depends on the pattern of fluorine substitution. The four F-PBDEs 25-4′F (5a), 27-4′F (5b); 69-4′F (5f), and 160-4′F (5i) where fluorine is substituted in the para position coelute with their corresponding parent PBDE. F-PBDE 1604′F, which has a higher degree of bromination in the nonfluorinated aromatic ring compared to F-PBDEs 25-4′F (5a), 27-4′F (5b), and 69-4′F (5f), can be partly separated, due to its rather long retention time. We found that there is a correlation between the ∆φ1 and ∆φ2 on one hand and the relative retention of the F-PBDE to its corresponding parent PBDE on the other hand. There is no correlation between the dipole moment and the retention time. The ∆φ caused by fluorine in the para position is the smallest, and the retention times are very similar, and the peaks show partial overlap. In the F-PBDE congeners 28-3′F (5c), 100-3F (5g), and 119-3F (5h), fluorine is substituted in the meta position and the increasing ∆φ induces lower boiling points and faster elution. The monofluorinated congener 28-3′F is well sepa3028

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rated; 100-3F and 119-3F show a small overlap with the corresponding parent PBDEs. This latter observation can be explained by the high degree of bromination of the fluorinated aromatic ring. The F-PBDEs 47-6F (5d) and 66-6F (5e) are ortho substituted by fluorine, and the highly increased ∆φ led to clearly lower boiling points. These F-PBDEs show up to 3 min shorter retention times. The fact that F-PBDEs can be separated from their corresponding parent compounds opens the way to use electron capture detection (ECD) instead of or in addition to MS. These results found in GC behavior support our hypothesis that F-PBDEs should show similar characteristics in many aspects to those of the corresponding nonfluorinated parent PBDEs. The ECNI mass spectra for F-PBDE 208-4′F and PBDE 208 are dominated by the bromine cluster m/z 486. This differs from the lower brominated F-PBDEs/PBDEs where the mass spectrum is dominated by the bromine ions (m/z 79, 81), Table 2. Interesting are the mass clusters at m/z 487 [M-C6-Br4-F], m/z 408 [M-C6Br5-F], and m/z 427 [M-C6-Br5] for F-PBDE 208-4′-F. The mass difference (m/z 19) between m/z 408 and m/z 427 is due to the additional fluorine. In EI mode, the mass spectra are dominated by the molecular ions. Proposed Analytical Application. F-PBDEs exhibit high potential as standards, markers, and tracers. Once incorporated into a substrate, fluorine functions as an indelible label that, with minimal background interference, can be used to monitor structures and dynamics, locating and quantifying significant products of chemical reactions and metabolic processes, which aid in understanding mechanisms of toxicology, chemical transport, degradation, and remediation. Aside from the analytical point of view, F-PBDEs can function as markers and tracers in mechanistic and toxicological studies and the information on electron distribution and sterical hindrance. In this respect, fluorinated analogues have distinct advantages: for instance, the van der Waals radii of fluorine and hydrogen are comparable, especially if C-F secondary binding (hyperconjugation) is taken into account. Therefore, the overall dimensions and the steric fit in enzyme receptor sites will not be greatly affected by fluorine substitution. The fluorine atom can block specific positions. In addition, fluorine changes the electronic distribution in the substrate without affecting the main values. Both, blocking and changes in the electron distribution in F-PBDEs in comparison to the corresponding parent PBDEs depend on the substitution pattern of the F-PBDEs. PBDEs in environmental and toxicological studies are often determined using GC-MS in ECNI mode, which is a very sensitive technique, but has the drawback that identification and quantification is primarily based on the 79 and 81 ions. The large advantage of F-PBDEs is that some of the fluorine congeners elute earlier than their parent PBDE analogues, and these compounds can, therefore, be used as appropriate internal standards.

Acknowledgments We would like to acknowledge Udo A. Th. Brinkman and Freek Ariese, Free University, Amsterdam, The Netherlands, Eric J. Reiner, Ministry of the Environment, Toronto, Canada, Jan Jensen, University of Iowa, and especially, Jan Scharp, Saxion University, Enschede, The Netherlands, and Gerd V. Ro¨schenthaler for stimulating discussions. In addition, we would like to thank the US EPA (RD-82902102) and the Alexander von Humboldt Foundation, Bonn, Germany, for financial support.

Supporting Information Available Table 3 containing 13C chemical shifts (δ) [ppm] relative to TMS) of F-PBDE congeners 25-4′F (5a), 47-6F (5d), 201-4′,6F

(5j), and 208-4′F (5k) in CDCl3 and DMSO-d6. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Received for review November 30, 2005. Revised manuscript received March 6, 2006. Accepted March 9, 2006. ES052410Z

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