BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. IS, 27-47 (1989)

A Comprehensive Gas Chromatographic/Mass Spectrometric Analysis of 4-Chlorobiphenyl Bacterial Degradation Products

Robert M d , t Frayois Messier, Christiane Ayotte, Marie-France Uvesque and Michel Sylvestre Institut National de la Recherche Scientifique (INRS-Santi), Universite du Quibec, 245, bod. Hymus, Pointe-Claire, Quebec H9R 1G6, Canada

Bacterial metabolism of khlorobiphenyl (dCB), a model compound of polychlorinated biphenyls, has been inves- tigated. Grown in the presence of 4-CB, Gram-negative strain B206 oxidized the noo-chlorinated ring to yield 2,3-dihydroxy-2,3ydrd-chlorobiphenyl, 3,4-dihydroxy-3,4dihydrd-chlorobiphenyl, as well as their corres- ponding 2,3 a d 3,4 catechol analogues, %hydroxy-4'-chlorobiphenyl and 4-hydroxy4'chlorobiphenyL The inter- mediate catechok were further oxidized to yield Zhydroxy-6-oxo-6-(4'-chloropbenyl)-bexa-2,4~ acid, Zhydroxy-&xo-(4'chlorophenyl)-hexanoic acid, 5-ox~y4'-chlorophenyl)-pentanoic acid, 4-ox0-4-(4'- chloropkny1)-butanoic acid, 4-chlorocinnamic acid and khlorobenzoic acid, which accumulates in the culture broths. The hydroxylated biotransformation products were characterized by gas chromatographic/mass spectro- metric analysis as trimethylsilyl (TMS) and d,-TMS derivatives, whereas metabolites with vicinal diols were a h analysed as their n-butylboronate derivatives. Gas chromatographic/mass spectrometric features of the metabolite derivatives are presented and 4-CB biodegradation pathways are discussed.

INTRODUCTION

Owing to the widespread contamination of environmental materials by polychlorinated biphenyls (PCBs),'.' there has been considerable interest during the past two decades in the fate of these ubiquitous pol- lutants since several of them have been shown to be carcinogenic in animal3 and mutagenic in bacterial model^.^ Investigations of the metabolism of pure indi- vidual PCBs in mammals indicated that hydroxylation to phenolic compounds, catalysed by hepatic micro- soma1 mixed function oxidase systems, is their major biotransformation route.' It has also been demon- strated that rate of metabolism of PCBs decreases as their degree of chlorination increases.6 Also, earlier studies by Jondorf et aL7 suggested that in addition to the degree of chlorination the position of the halogen atoms on the biphenyl molecule is an important factor which greatly influences metabolic rates. A study by Schulte and Acker' with 20 PCB congeners ranging from tetra- to octachlorobiphenyls clearly revealed the principle that PCBs are metabolized if there is at least one pair of adjacent, unsubstituted carbon atoms.

The bacterial biodegradation of PCBs is also influ- enced by substitution patterns of chlorine atoms on their aromatic rings. It was shown by several investiga- tors that biodegradation rates of PCBs decrease as chlorine substitution i n c r e a ~ e s . ' ~ ~ ~ ' ~ The position of chlorine atoms is also an important factor determining the bacterial degradation of PCB congeners.' ','' Ahmed and Fochti3 reported that chlorinated benzoic acids are produced as a result of oxidative degradation

t Author to whom correspondence should be addressed

0887-6 1 34/89/01OO27-2 1 $10.50 0 1989 by John Wiley & Sons, Ltd.

of mono- and dichlorobiphenyls by two species of Achromobacter. It was originally proposed by Gibson et u I . ' ~ * ' ~ that in bacteria aromatic compounds are con- verted through cyclic peroxide intermediates to dihy- drodiols and ultimately to catechol derivatives via a dioxygenase-catalysed reaction.

Studies with various microorganisms indicated that biphenyl' 391'-17 and low chlorinated biphenyl' '*''-'' catechol metabolites are further transformed to a- hydroxy-y-phenylmuconic semialdehyde and benzoic acid and to 2-hydroxy-6-oxo-6-(chlorophenyl)-hexa-2,4- dienoic acid and chlorinated benzoic acids, respectively, via meta-type oxidative cleavage.

Dihydroxylation of the biphenyl ring in the C2 and C, positions appears to be the predominant hydroxyl- ation mechanism for bacterial degradation of PCBs. However, it was recently suggested that the ability of Alcaligenes eutrophus H850 to degrade some of the ortho-substituted PCB congeners be attributed to its ability to hydroxylate these molecules in positions C, and C4 ." However, a 3,Chydroxylation was only indi- rectly demonstrated, mainly on the basis of PCB con- geners' relative biodegradabilities.

The bacterial strain B-206 used in this study was originally described as being able to grow on 4- chlorobiphenyl(4-CB) and degrade this substrate,23 and also to have the remarkable property to transform 4-CB into its corresponding isomeric 2-hydroxy-3-nitro and 4-hydroxy-3-nitro derivative^.'^ Lately, we reported that 4-CB was metabolized by Achromobacter sp. (B- 218) and Bacillus breuis (B-257) strains, yielding 4- chlorobenzoic acid (CCBA) as a major biodegradation product.'' Our rationale for investigating the microbial degradation of 4-CB was based on the fact that the chlorine atom in this model compound of PCBs is situ-

Received 14 April 1988 Accepted 7 July 1988

28 R. MAS!& ET AL.

ated at a metabolically strategic position and also on the fact that 4-CB possesses an unsubstituted aromatic ring which is vulnerable to microbial metabolism. A primary objective was to identify 4-CB biodegradation products so as to characterize its major metabolic path- ways in selected microorganisms. A second objective was to provide reference mass spectral data as evidence for the mechanisms of 4-CB metabolism in strain B-206. We report in this paper the isolation and gas chromatographic/mass spectrometric characterization of 4-CB biodegradation products by Gram-negative strain B-206. Mass spectrometric data demonstrating both 2,3- and 3,4-dihydroxylation of 4-CB are presented for the first time.

EXPERIMENTAL

Chemicals

Ethyl acetate, acetonitrile and pyridine were redistilled prior to use. Silylating reagent, N-O-bis-(trimethylsily1)- trifluoroacetamide (BSTFA) was obtained from Pierce Chemical Co. (Rock ford, Illinois) and N-O-bis- (trimethyl-d,-si1yl)acetamide (deuterated BSA) from Merck Sharp and Dohme Isotopes (Pointe-Claire, Quebec, Canada). Acetic anhydride was obtained from Applied Science (State College, Pennsylvania). N-Butyl boronic acid (Aldrich Chemical Co., Milwaukee, Wisconsin) was recrystallized in toluene before use. 4-CB was purchased from Aldrich Chemical Co. and its purity (99.9%) was assessed by gas chromatography/ mass spectrometry (GC/MS).

Organism and growth conditions

Strain B-206, a Gram-negative bacterium, was isolated from an activated-sludge samplez3 and was kept lyophi- lized in bovine serum until use. Its taxonomic character- ization has already been reported.23 The organism was grown in a basal mineral medium (pH 7.2) whose com- position has been previously reported.23 Trace elements and EDTA were supplied by the addition of 0.5 ml 1-' of the 'metal 44' formulation described by Cohen-Bazire et a1.26

The bacterium was grown as already described,23 and the cell concentrations were adjusted so as to obtain about lo9 cells ml- of medium. 4-Chlorobiphenyl (50 mg in 1 ml diethyl ether) was added to 125 ml Erlen- meyer flasks containing 30 ml of freshly grown cell sus- pensions. The mixtures were incubated in the dark at 29°C for various periods of time (24, 48, 96 and 144 h) in a shaking incubator operating at 250 rev/min under a controlled atmosphere containing 10% carbon dioxide in air. No non-bacterial degradation products were detected in control broths, where 4-CB was incubated in the absence of the microorganism.

Isolation of the metabolites

After appropriate incubation times, the bacterial broths were filtered through a Whatman paper no. 40 in order

to recover residual and unchanged 4-CB which did not solubilize during incubation. The bacterial cells were pelleted by centrifugation (SO00 rev/min for 10 min) and removed. The supernatants were extracted with three volumes of ethyl acetate. The solvent was evaporated to dryness at 40°C under a stream of nitrogen and the residues dissolved in 1 ml ethyl acetate. These solutions were kept in the dark at -20°C until analysed. The aqueous phases were acidified with 1.0 M HC1 to pH 3 and processed as described above.

Chemical derivatization

Trimethylsilyl (TMS) and ('H,)TMS derivatives. A 50 p1 aliquot of an ethyl acetate extract was evaporated to dryness under a stream of nitrogen at 40 "C. The residue was dissolved in a mixture of BSTFA or deuterated BSA (10 p1) and acetonitrile (40 pl), and heated at 70°C for 15 min. A 1 p1 aliquot of this solution was injected into the chromatograph for GC and GC/MS analysis.

n-Butyl boronate derivatives. A 50 pl aliquot of an ethyl acetate extract was evaporated to dryness under a stream of nitrogen at 40 "C and the residue dissolved in 50 pl of dry ethyl acetate. Recrystallized n-butyl boronic acid (10 mg) dissolved in 50 p1 ethyl acetate was added and the mixture heated at 70°C for 15 min; 1 pl was injected for GC/MS analysis.

GC

Gas chromatography was carried out with a Perkin Elmer Model Sigma 2 gas chromatograph, equipped with a split/splitless injector, flame ionization detector and an open fused-silica capillary column (25 m x 0.2 mm i.d.) wall coated with SE-30 (Chromatographic Specialities, Brockville, Ontario, Canada). Helium (1 ml min-') was used as carrier gas. The samples were injected in the splitless mode. The initial oven tem- perature (140 "C) was held for 5 min and programmed at 5°C min-' to 290°C and kept for 15 min. The injec- tor and the detector temperatures were maintained at 250 "C and 300 "C, respectively.

GC/MS

The mass spectra were recorded on a Kratos MS-25 mass spectrometer equipped with a Perkin Elmer Sigma 3 gas chromatograph. The gas chromatograph was equipped with a capillary column similar to that described above, and the column was introduced directly into the mass spectrometer ion source. The injector, interface and ion source temperatures were maintained at 250 "C. The mass spectrometer was oper- ated in the electron ionization (EI) mode (70 eV) at an acceleration voltage of 4 kV and at a resolution of lo00 @/Am 10% valley). The mass range of 50-600 u was repetitively scanned at a rate of 1 s per decade.

High-performance liquid chromatography (HPLC)

A Perkin Elmer model 601 liquid chromatograph equipped with a Magnum 9 reversed-phase column

BACTERIAL DEGRADATION PRODUCTIONS 29

(9.2 mm id. x 25 cm) (Mandel Scientific Company, Montreal) and a Perkin Elmer LC-55 ultraviolet-visible spectrophotometer operated at 254 nm were used. Sol- vents used were A: 32% MeOH/ACN and B: H,O. A 60 min gradient was run from 35% A to 100% A at a flow rate of 1.5 ml min-'. Aqueous fractions corres- ponding to the chromatographic peaks were collected and extracted with ethyl acetate. The organic extracts were processed as described above and analysed by GC/MS.

first group is that pertaining to metabolites resulting from regiospecific hydroxylation reactions of the non- substituted aromatic ring of 4-CB. This group com- prises the 'phenolic metabolites'. The metabolites bearing a carboxylic acid function compose the second group of 4-CB biodegradation products. They result from further oxidative metabolism of some of the inter- mediate phenolic metabolites. The chromatographic properties and the molecular ions of the TMS and d,-TMS derivatives of 4-CB bacterial metabolites are summarized in Table 1.

~ ~ ~ ~~~

RESULTS AND DISCUSSION ~ ~~ ~ _ _ _ _ ~

Extraction of the bacterial broths was first carried out at neutral pH to isolate phenolic compounds and to prevent the degradation of metabolites which could be unstable under acidic conditions. Evaporation of the ethyl acetate acidic extracts (pH 3) afforded an abun- dant white precipitate identified as 4-chlorobenzoic acid (1) (4-CBA) by GC/MS of its TMS (M", 228) and d,-TMS (M", 237) derivatives and comparison with authentic 4-CBA. T h s acidic metabolite accounted for more than 50% of all ethyl acetate soluble metabolites. For quantitative data regarding 4-CBA production from the bacterial degradation of 4CB, readers should refer to our previous paper^.^^,^' As one will notice further, all 4-CB metabolites bear the chlorophenyl moiety of the parent substrate. This illustrates the natural propensity of most microorganisms to metabo- lize preferentially natural and non-substituted aromatic compounds. This observation is also in accordance with previously reported data of the microbial degradation of biphenyl and chlorinated biphenyl^.'^-^^^^^^^^

Preliminary GC/MS analysis of the crude bacterial extracts indicated that the other biodegradation pro- ducts of 4-CB can be grouped into two distinct groups according to their characteristic functional groups. The

Identification of the phenolic metabolites

The crude and neutral ethyl acetate extracts were puri- fied by HPLC in order to separate the phenolic com- pounds from some of the coextracted acidic metabolites. The phenolic metabolite containing HPLC fractions were pooled and derivatized with BSTFA. Figure 1 shows a characteristic GC/MS profile of 4-CB phenolic metabolites obtained after 96 h incubation with strain

Metabolite 3 and 5 TMS derivatives (Fig. 2) both gave mass spectra (M", 276) with similar ion fragmen- tation patterns. The mass shift of 9 u from the molecu- lar ion to m/z 285 is indicative of monohydroxylated metabolites (Fig. 2(b) and (d)). In addition, comparison of the ratio and the intensity of m/z 93 and 95 fragments indicated the presence of an 0-TMS group, which is in closer proximity to the chlorine atom in compound 3 than in 5.'' These ions were shifted to m/z 99 and 101 in the mass spectra of their d,-TMS derivatives. They arise from the migration of the chlorine atom on the TMS group with subsequent rearrangement of the intermediate silyl group prior to fragmentation. The resulting ion probably has the structure [Me,-Si-Cl] +. Mass spectral migration of various groups to silicon is not an unusual phenomenon in

B-206.

Table 1. Gas chromatographic retention and prtial mass spectral features of 4-CB bacterial metabolites

Compound'

1 4-Chlorobenzoic acid 2 4-Chlorocinnamic acid 3 2-Hydroxy-4'-chlorobiphenyl 4 4-Chlorobenzoyl propionic acid 5 4-Hydroxy-4'-chlorobiphenyl 6 4-Chlorobenzoyl butanoic acid 7a 2,3-Dihydroxy-2,3-dihydro-4'-chlorobiphenyIc 7b 3.4- Dihydroxy-3.4-di hydro-4'-chlorobiphenylC 8a 2,3-Dihydroxy-4'-chlorobiphenyl' 9 6-Hydroxy-6-(4-chlorophenyl)-hexanoic acid

10 2-Hydroxy-6-oxo-6-(4'-chlorophenyl)-hex-2,4-dienoic acid

11 2-Hydroxy-6-oxo-6-(4'-chlorophenyl)- hexanoic acid 12 2-Hydroxy-6-oxo-6-(4'-chlorophenyl)-hex-4-enoic acid

Mi '

RRTa TMS d,-TMS

1 .oo 1.54 1.74 1.91 2.1 4 2.25 2.27 2.28 2.37 2.42

a 2.84 b 2.87 c 3.02

3.1 1 3.1 4

228 254 276 284 276 298 366 366 364 386 396 396 396 400 398

237 263 285 293

307

384

404 41 4 41 4 41 4 41 8 41 6

285

384

382

"Compound numbers correspond to labelled peaks in Figs 1 and 7.

' Relative retention times of the n-butyl boronic ester derivatives were: 2.57 for the coeluting dihydrodiols 7a and 7b and 2.65 for the catechol 8a.

Retention time relative to that of 4-chlorobenzoic acid TMS derivative, 18.1 5 rnin.

30 R. MAS& ET AL.

100

90

90

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68

50

40

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10

I 2

1 58 100 200

5 -

,:I 250

b

8a

scan number Figure 1. Typical reconstructed TIC chromatogram from the GC/MS analysis of 4-CB phenolic metabolites extracted at neutral pH from a culture medium obtained after 96 h incubation of 4-CB with strain 8-206. The extract was purified by HPLC and derivatized prior to analysis. See Table 1 and text for analytical conditions and identities of labelled peaks.

mass spectrometry of silicon compounds and has been reported by several author^.*^-^'

Since mass spectral data indicate that hydroxylation occurs on the non-substituted ring of 4-CB, compounds 3 and 5 were assigned the structures 2-hydroxy-4-chlo- robiphenyl and 4-hydroxy-4-chlorobiphenyl, respec- tively. The proposed structures are further ascertained by their chromatographic retention, since both com- pounds 3 and 5 elute in the same order as 2-hydroxy-4'- nitrobiphenyl and 4-hydro~y-4-nitrobiphenyl.~~ Fur- thermore, it was reported in the literature that ortho- hydr~xybiphenyl~~ and ortho-chlorophenol'4 elute before their para-substituted analogues in GC on the column coating (SE-30) used in this study. Since no ref- erence compounds were available, there is no irrefutable evidence that compound 5 is not the meta- rather than the para-substituted hydroxychlorobiphenyl. However, comparison of the chromatographic retention and mass spectra of compound 5 and its TMS derivative with those of compound 3, 2-, 3- and 4-hydroxybiphenyl, 4-CB and biphenyl strongly indicates that compound 5 bears a 4-hydroxy substituent. The formation of mono-

hydroxylated polychlorinated biphenyl has never been reported as an important biotransformation pathway of the PCB congeners, whose metabolism was investigated in bacteria.'s*20,21 In previous publications, we re- ported that isomeric monohydroxylated metabolites of chloro- and nitro-substituted biphenyl can be produced in relatively significant amounts in b a ~ t e r i a . ~ ~ . ~ ~

The formation of the monohydroxylated 4-CB isomers 3 and 5 can be rationalized on the basis of dehydration reactions of 2,3- and 3,4-dihydrodiol intermediate^^^.'^ (Scheme 3). The formation of these compounds from 4-CB will be further discussed later in the text.

The abundant metabolites 7a and 7b eluting as a par- tially resolved doublet centred at 18.45 min in the total ion current (TIC) chromatogram shown in Fig. 1 both showed a parent ion at rn/z 366 (Figs 3 and 4). The mass spectral fragmentation patterns of their TMS deriv- atives were very similar and were characterized by ion fragments at m/z 351 [M - 15]+, 276 [M - go]', 261 [M - 15 - 901' and 93 [Me,Si=Cl]+. The mass spec- tral properties of the corresponding d9-TMS derivative

BACTERIAL DEGRADATION PRODUCTIONS

267

139 168

31

L

100 r3 a I , , , , l ~ L . i . J ~ , , , , 1 , , , , , ,

0 50 100 1 5 0 2 0 0 2 5 0 30 0 350 400

100

0

100

0

ioa

c

b

C

152

? , , , ( ' 5 0 4 & h 1 0 0 1 5 0 200 2 5 0

73 93

mlz

d

99 82 I 133 152

'' Mt 276

Figure 2. Mass spectra (GC/MS El) of Z-hydroxy-4'-chlorobiphenyl (3) (a and b) and 4-hydroxy-4'-chlorobiphenyl (5) (c and d) as TMS and d,-TMS derivatives.

32 R. MASSk ET AL.

66

50

40

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0

lo@ 1, B

3 c,*Ms

\ / \ / - 7a

93

M+' 366 147

276 1 100 150 250 300 356

/

82

99 162

I

50 100 150 200 250 300 350

m / z Figure 3. Mass spectra (GC/MS El) of 2.3-dihydroxy-2,3-dihydro-4-chlorobipheny~ (7a) as TMS (a) and d,-TMS (b) derivatives.

BACTERIAL DEGRADATION PRODUCTIONS 33

60

50

40

30

20

I B

0

3 lj,OTMS M+'

366

147

351

100 150 250 300 350 L

0& 50

I2 H ,O-~QTMS

M+. 384

162

366

285

100 150 200 250 300 350 I ' I I I I I J

m / z Figure 4. Mass spectra (GC/MS El) of 3,4-dihydroxy-3.4-dihydro-4'-chlorobiphenyl (7b) as TMS (a) and d,-TMS (b) derivatives.

34 R. MASSI? ET AL.

l+' . , r n M S - L TMSOH

- 7a

M+' 366 M+' 384[dgTMS]

rn/z 276 m/Z 285 [d~-TMS]

1 L .

Me, ,Me/' l+. TMSO OTMS

- Me@ .,& L 8a -

M+' 364 M+ ' 382[dg -T MS]

m/z 276 rn/z 282[dg-TMS]

Scheme 1. Proposed structures for ions at m/z 276 in the mass spectra of compounds 7a. 7b and 8a TMS derivatives.

(Figs 3(b) and 4(b)) indicate the presence of two hydroxyl groups in compounds 7a and 7b, the molecu- lar ions being shifted by 18 u to m/z 384. The data also showed that the ion at m/z 276 arises from the elimi- nation of one molecule of trimethylsilanol (TMSOH) from the molecular ion, indicating the presence of a dihydrodiol moiety in both compounds 7a and 7b (Scheme 1).

The respective position of the isomeric dihydrodiol group in compounds 7a and 7b was assigned on the basis of the above-mentioned mass spectral data and by comparison of their retention times with that of chlorin- ated catechol. Generally, 2,3-hydroxychlorophenols elute before 3,4-dihydroxychlorophenol~.~~ One may expect the same trend for the elution order of the dihy- drodiols 7a and 7b. As expected, the first eluted dihy- drodiol, 7a, showed a structurally informative ionic doublet at mfz 93, 95 which was much more intense than that in compound 7b's mass spectrum. This obser- vation is in accordance with the mass spectral data of the isomeric monohydroxy TMS derivatives 3 and 5 discussed above. Compounds 7a and 7b were respec- tively assigned the structures 2,3-dihydroxy-2,3- dihydro-4-chlorobiphenyl and 3,4-dihydroxy-3,4- dihydro-4-chlorobiphenyl.

The mass spectrum of compound 8a TMS derivative (Fig. 5(a)) was characterized by a molecular ion at m/z 364 and also by the formation of a structurally informa- tive ion at m/z 276, which was shifted by 6 u to m/z 282 in the mass spectrum of its d9-TMS derivative (Fig. 5(b)). This ion, which arises from the loss of one mol- ecule of tetramethylsilane from the molecular ion [M - Me,Si]+, is indicative of the presence of a cate- chol m~iety.~ ' .~ ' As shown in Scheme 1, the rearrange- ment of the vicinal OTMS groups leads to the formation of a cyclic ion at m/z 276 containing a silyl atom bridged by the ortho oxygen atoms. Although the TMS derivatives of compounds 7a, 7b and 8a give rise to a common ion at m/z 276, TMS-deuterium labelling was used here with advantage to differentiate their respective structural features. Compound 8a was assign-

ed the structure of 2,3-dihydroxy-4-chlorobiphenyl on the basis of the mass spectral data presented above.

The formation of cyclic n-butylboronates from com- pounds 7a, 7b and 8a provided further mass spectral evidence to distinguish unambiguously between cate- chol and dihydrodiol metabolites of 4-CB. Their n- butylboronate derivatives were prepared by treatment of the metabolites (purified by HPLC) with n- butylboronic acid. The dihydrodiols 7a and 7b, which were barely chromatographically resolved as TMS derivatives (Fig. I), were not further separated by GC/MS analysis of their n-butylboronate derivatives and eluted as a single peak (Table 1). The resulting mass spectrum is shown in Fig. qa). The n-butylboronate derivatives of the dihydrodiols 7a and 7b are extensively fragmented upon EI shown in Scheme 2.

The fragmentation is initiated by the loss of a chlor- ine radical to yield the ion of m/z 253, whereas the elimination of the n-butyl group from M" gives rise to the cyclic oxonium-type ion of m/z 231. EI also induced the loss of the neutral C,H,BO moiety from the molec- ular ion to yield the keto form of a 2- or 3-hydroxy-4- chlorobiphenyl type ion at mfz 204 (the corresponding 3-keto form is shown in Scheme 2). The latter ion loses successively CO and Cl', producing the m/z 176 and 141 fragments, respectively, whereas direct loss of C1' gives rise to the fragment of m/z 169. In a similar fragmenta- tion pathway, the molecular ion loses the neutral n-C,H,BO, moiety and one molecule of HCl to yield the abundant ions of m/z 188 and 152, respectively.

The major fragmentation sequence in the mass spec- trum of compound 8a (Fig. qb)) first involved the loss of an n-butyl moiety from the molecular ion with sub- sequent elimination of hydrochloric acid to yield an abundant ion at m/z 230 ([M - 56]+) and an ion of low abundance at m/z 194. Other minor fragment ions at m/z 204,188,169,167 and 152 (most of which are abun- dant fragment ions in the mass spectra of compounds 7a and 7b) are likely to arise from fragmentation path- ways analogous to those prevailing for compounds 7a and 7b n-butylboronate derivatives (Scheme 2).

BACTERIAL DEGRADATION PRODUCTIONS

TMSO OTMS

35

M +. 261 147 246 276 359

1

382 246 264282

I#,

I II

1

82

162

\ rnl 1 1 0 Ill1 inn 170 1-40 160 I00 200

M/Z Figure 5. Mass spectra (GC/MS El) of 2,3-dihydroxy-4'-chlorobiphenyl (8a) as TMS (a) and d,-TMS (b) derivatives.

90

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78

68

50

40

38

20

I0

0

100

90

80

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60

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48

38

20

I0

0

A 9 4 " 9 B

0' '0

7a -

75

115 1

I I

50 I 00

B

141 176 152

169

;J 150

m l

" C4"9 B

0' '0

204

6

190

- r J 231

M- 28

253

200 250

8a

230

139

75

M-f 286

m l z Figure 6. Mass spectra (GC/MS El) of 2,3-dihydroxy-2,3-dihydro-4'-chlorobiphenyl (7a and 7b) (a) and 3,4-dihydroxy-4'-chlorobi- phenyl (8a) (b) as n-butyl boronic ester derivatives.

BACTERIAL DEGRADATION PRODUCTIONS 37

WBU I

m/z 253

mb 176

m/z 141

CI' - - co - -

__h_

R M+' 288

BUBO t

I-"' Scheme 2. Major fragmentation pathways of compounds 7a and 7b n-butyl boronic ester derivative under El ionization.

It is noteworthy that only the 2,3-catechol metabolite 8a accumulated in the bacterial broths, although both the 2,3 and 3,4-dihydrodiols 7a and 7b, their immediate metabolic precursors, were isolated and characterized. Additional experiments with strain B-206 did not reveal the presence of the 3,4-catechol 8b (Scheme 3) although its immediate metabolic precursor, the 3,4-dihydrodiol 7b, was repeatedly detected in the bacterial cultures. This is the first time that mass spectrometric data are reported as evidence for the 3,4-dihydroxylation of 4-CB in bacteria.

The kinetics of their formation and transformation into compounds 7a and 7b were not investigated in this study, but their formation was proposed on the basis of the data presented above and that reported previously in the l i t e r a t ~ r e . ' ~ - ~ ~ . ~ ~ ~ ~

However, we reported in earlier studies with strain B-206, where 4-nitrobiphenyl was used as substrate, the GC/MS identification of 3,4-dihydroxy-3,4-dihydro-4- nitr0bipheny1.j~

The data presented above suggest that the initial step in the bacterial degradation of 4-CB is, as is generally

Scheme 3. Proposed metabolic pathway for the formation of dihydrodiols 7a and 7b and catechols 8a and 8b in 4-chlorobiphenyl bac- terial metabolism.

38 R. MASSfi ET AL.

accepted, a dioxygenase-catalysed reaction3943 that leads to the formation of two isomeric cis-dioxetanes through the addition of two atoms of molecular oxygen at the 2,3 and 3,4 positions on the non-chlorinated ring of 4-CB (Scheme 3). The resulting cyclic peroxide inter- mediates are apparently converted rapidly to the corres- ponding 2,3- and 3,rl-dihydrodiol 7a and 7b since they were not detected in the bacterial broths. Enzymatic catalysed dehydrogenation of the latter dihydrodiols yield the catechols 8a and 8b. However, compound 8a was the sole chlorocatechol detected by GC/MS analysis of the bacterial broths. This observation indi- cates that the isomeric chlorocatechol 8b is probably produced in smaller amounts and/or degraded by further oxidative metabolism at a faster rate than its analogue 8a by strain B-206. This particular aspect of 4-CB metabolism by strain B-206 justifies further inves- tigation since it was recently suggested that chlorobi- phenyl metabolism is extended to a larger number of congeners in bacterial strains that have either 2,3- and/or 3,4-dio~ygenases.~~ The rate of dihydroxylation

100

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0

reactions in one of the two alternative positions would then influence the ability of the strain to degrade a larger number of PCB congeners. Owing to their pro- pensity to form more stable aromatic compounds, the dihydrodiols 7a and 7b undergo elimination of one mol- ecule of water to give selectively the corresponding monohydroxylated compounds 3 and 5, respectively, since no trace of 3-hydroxy-4-chlorobiphenyl was detected in the culture media. The strong preference for the formation of only two of the three possible phenolic isomers is frequently observed, and predictable by molecular orbital theoretical method^?^,^'

Identification of the acidic metabolites

The reconstructed TIC chromatogram of the ethyl acetate extractable acidic metabolites formed after 96 h of incubation of 4-CB with strain B-206 is shown in Fig. 7. The metabolites were purified by HPLC prior to GC/MS analysis. The proposed formation of the major

100 150 280 250 300 350

scan number

n

L2

L 480 450

Figure 7. Typical reconstructed TIC chromatogram from the GC/MS analysis of 4-CB acidic metabolites extracted at pH 3.0 from a culture medium obtained after 96 h incubation of 4 - C B with strain 8-206. The extract was purified by HPLC and derivatized prior to analysis. See Table 1 and text for analytical conditions and identities of labelled peaks.

BACTERIAL DEGRADATION PRODUCTIONS 39

2 - 6 - Scheme 4. Proposed pathway for the formation of some 4-CB acidic metabolites by 1,2-rnete cleavage of catechol 8a and proposed structure of metabolites-arising from secondary metabolic routes.

acid metabolites arising from oxidative cleavage of com- pound 8a is outlined in Scheme 4. The compounds con- tained in chromatographic peaks lOa, 10b and 1Oc (Fig. 7) showed identical mass spectral properties, which are illustrated in the mass spectrum of compound 10b TMS derivative in Fig. 8(a). The latter metabolite is charac- terized by low-abundance molecular and [M - 151' ions at m/z 396 and 381 and by a prominent ion at m/z 279 [M - 1171' arising from the loss of the 'COOTMS group from the molecular ion. The mass spectrum of the corresponding d,-TMS derivative (Fig. 8(b)) shows an increase of 18 u of the molecular ion at m/z 414, indicating the presence of two hydroxyl groups in com- pounds 10a, 10b and 1Oc. The elimination of the 'COOTMS group leads to the formation of a promi- nent oxonium-type ion at mJz 279 which retains one OTMS group (m/z 288, d,-TMS). The high stability of this ion arises from the conjugation of the double bond of the oxonium ion with those of the unsaturated chain, and also with the chlorobenzoyl moiety. The fragment ion corresponding to the chlorobenzoyl moiety which one would have expected at m/z 139 is virtually absent in the mass spectra of these isomeric acids. This mass spectral feature is characteristic not only of these acidic metabolites but also of their corresponding saturated and monounsaturated analogues 11 and 12, as will be discussed below. The mass spectral features of com- pounds lOa, 10b and 1Oc are in accordance with those of the TMS derivatives of analogous unsaturated acids obtained from the bacterial degradation of some poly-

chlorinated biphenyl congeners.'8*20*2' Thu s, they were identified as isomers of 2-hydroxy-6-oxo-6-(4-chloro- phenyl)-hexa-2,4-dienoic acid. As illustrated in Scheme 4, this acid arises from the oxidative metu cleavage occurring at the 1,2 positions of 2,3-dihydroxy-4'-chlo- robiphenyl 8a.18-21,25

The formation of three isomers of compound 10 occurs probably during the oxidative 1,Zmeta cleavage of catechol Sa, where the formation of the C,-keto and C,-carboxylic functions directs the delocalized double bonds of the catechol nucleus at the C2 and C, posi- tions in the oxidation product 10, thus promoting iso- merization of the double bonds. Alternatively, the two double bonds can readily isomerize by enolization at C , and C, in the bacterial broths. The relative amounts of isomers 1Oa-c were fairly constant from one experiment to another, indicating that an equilibrium state is rapidly reached between the isomeric forms as com- pound 10 is produced in the culture medium.

The mass spectra of the TMS and d,-TMS deriv- atives of the saturated acid 11 are compared in Fig. 9. The presence of two hydroxyl functions was ascertained by the 18 u shift of the molecular ion from m/z 400 (Fig. 9(a)) to m/z 418 (Fig. 9(b)). Strong evidence for the struc- tural features of this metabolite was provided by the fragment ion at m/z 283 (m/z 292, d,-TMS) which arises from the loss of the terminal 'COOTMS radical from the molecular ion. The presence of a methylene group in a &position to the carbonyl group favours the selective formation of the prominent m/z 129 ion (m/z 138,

I OH

'111

1111

ill

1.11

1.11

,111

111

,*I1

111

I 1

A

1.11

279 C l e C - C H = C H - C H = C i -COOTMS

TMSO II 0

-

10a-c

73

I1 47

l G l l I00 200 z20 11111 1711 1411

208

11111

'Ill

I l l 1

i l l

1.11

!,I1

.Ill

.!I1

;I1

I II

2

1162 1189

.III mi 110 i i in 17n I.III im iun zoo 220

MIZ Figure 8. Mass spectra (GC/MS El) of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)-hexa-2,4-dienoic acid (lob) as TMS (a) and d,-TMS (b) derivatives.

I 00

90

90

70

60

58

40

30

20

18

0

I 08

90

a0

70

60

50

40

30 -

20

10

0

A

73

B

,Ibl 50

I 00

129

C I O C-CH,-CH,-CH,-CH-COOTMS I

0 TMSO - I t

11 -

147

283

2

111

A 100

150 200 ise 308 350 400

138

292

162

150 20a 250 300 350 408

MIZ Figure 9. Mass spectra (GC/MS El) of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)-hexanoic acid (11 ) as TMS (a) and 13,-TMS (b) derivatives.

42 R. MASS6 ET AL.

C I e C - [ C H ,I3- YH-COOTMS II

- 0 ~ O T M S

M+' 400

Me:/ M+' 4 18 [dg-TMS]

m/z 38s

R\ ,w\ C L CH, I t I + o ~CH-CH=OTMS

H' m/z 283 m/z 292[dgTMS]

+ + CH,=CH-CH=OlMS

m/z 129

m/z 138[dg-TMS]

$. -co

R\ C /c2\CH, I1 I + + 0 n P H - C H =0-SiMe,-OTMS .) CH ,=CH-CH=O-SiMe,-OTMS

H m/z 357 m/z 203

rn/Z 3 72 [4-T M S] m/Z 2 18 cd9-T M S]

Scheme 5. Formation of the prominent ion of m/z 129 and structurally informative ions of m/z 357, 283 and 203 in the mass spectrum of compound 11 TMS derivative.

dg-TMS) through a McLafferty rearrangement of the ion of m/z 283, as shown in Scheme 5. A minor but mechanistically interesting process in the fragmentation of the cyclic oxonium [M - 151' ion is the m/z 385-357 transition, which involves the neutral loss of

CH "o+

I J C I m/z 357 H q,SiMe,

R JMS

A SiMe, 0' f M S

$. -TMSOH

+ R-CH=CH-CeC-O=SiMe,

m/z 263

Scheme 6. Formation of ions at m/z 263.267 and 265 in cornpou nds 1 OH, 11 and 12, respectively.

one molecule of CO to yield the intermediate ion at m/z 357 (m/z 372, dg-TMS). A similar fragmentation pathway was observed for a number of a-monohydroxy acid TMS derivatives, where the [M - Me - CO]' ion is also a characteristic mass spectral The latter fragment ion rearranges through a McLafferty rearrangement to give an ion at m/z 203 (m/z 218, d,-TMS) (Scheme 5). Minor but structurally informative ions at m/z 139 (chlorobenzoyl moiety), m/z 310 (M - TMSOH; m/z 319, d,-TMS) and m/z 267 (m/z 273, I,-TMS) provide complementary evidence for com- pound 11 structural features. The structure of the ion at m/z 267 is proposed in Scheme 6, along with those of ions of similar origin and arising from fragmentation of the acidic metabolites 1Oa-c (Fig. 8) and 12 (Fig. 10).

The mass spectral features of compound 12 TMS derivative (Fig. 10) are very similar to those exhibited by the isomeric diunsaturated acids 1Oa-c. The molecu- lar ion at m/z 398 (m/z 416, d,-TMS) indicates the pre- sence of only one double bond in the carboxylic acid chain. It is noteworthy that compound 12, which under- goes an initial fragmentation (loss of a 'COOTMS radical) identical to that of compounds 1Oa-c and 11 (Figs 8 and 9) also loses a hydrogen radical to yield a characteristic ionic cluster at m/z 280-283. The elimi- nation of a hydrogen radical is likely to occur from the M - COOTMS ion at m/z 281, yielding the odd- electron ion of m/z 280. Furthermore, it is very unlikely that the double bond is located at C, , since the forma- tion of the ion at m/z 280 would then require both the migration of one hydrogen atom from C, or C , to C, with subsequent elimination of 'H.

70 -

60 .

50

4n .

30 . 280 I

1281

12 -

240 200 380 328 348 368

73

147 139 I

191 h h B e I on I 2 8 I40 I60 188 208 220

289 [2w 317 398 M+

41 6 b l m I I I I I I I 1 6

243 268 200 3Ufl 328 348 368 388 488 420

ly/ 7(1 r2 31, - 1 I I I

6 8 00 I ao 128 I 48 160 188 288 228 243-

Ml z Figure 10. Mass spectra (GC/MS El) of 2-hydroxy-6-oxo-6-(4'-chlorophenyl)-hex-4-enoic acid (12) as TMS (a) and d,-TMS (b) deriv- atives.

44 R. MAS!& ET AL.

213 * T + 173 A

I 213 I

H,- C H;-C H ,-C H,-COOT M S

- 9

[M-15]+ 371

2 111 21.11 2110 JIW

73

173 93

17117rrrrllllllrT- I on 2 0 0

82

1182

M/Z Figure 11. Mass spectra (GC/MS El) of 6-hydroxy-6-(4'-chlorophenyl)-hexanoic acid (9) as TMS (a) and d,-TMS (b) derivatives.

BACTERIAL DEGRADATION PRODUCTIONS 45

Additional evidence for the presence of a double bond at the C, position in compound 12 was provided by the formation of a low-abundance but highly struc- turally informative chlorine containing ion at m/z 265 (m/z 271, d,-TMS). Interestingly, the diunsaturated and saturated acidic metabolites 1Oa-c and 11 yield struc- turally related ions at m/z 263 (m/z 271, d,-TMS) (Fig. 8) and at m/z 267 (m/z 273, d,-TMS) (Fig. 9). In the latter figure, the ion of m/z 267 arises from the elimi- nation of one molecule of TMSOH from the ion of m/z 357 [M+' -Me -CO] (Scheme 5 ) according to the mechanism proposed in Scheme 6.

Although the [M - Me - CO]' ion is not detected in the mass spectra of compound 10a-c and 12 TMS derivatives, the genesis of ions of m/z 263 and 265 is likely to occur following a fragmentation pathway similar to that proposed for the formation of the ion of m/z 267 in compound 11 TMS derivative (Scheme 6).

Although their route of formation has never been extensively investigated, compounds 11 and 12 are likely to originate from the enzymatic reduction of isomeric diunsaturated acids 1Oa-c as shown in Scheme 4. This is supported by the fact that catalytic hydro- genation of the unsaturated acids 10 and 12 on plati- num oxide (PtO,/H,) afforded the saturated acid 11.

The compound corresponding to peak 9 was assigned the structure of 6-hydroxy-6-(4'-chlorophenyl)-hexanoic acid on the basis of the characteristic mass spectra of its TMS and d9-TMS derivatives (Fig. 11). Its structural features were characterized by the presence of ions of m/z 173 and 213 resulting from the cleavage of the C5-C, bond and by low-abundance ions at m/z 239 and 253 (Scheme 7). The ion of m/z 239 originates from a McLafferty rearrangement of the [M - 15]+ ion involving the keto group of the carboxylic acid function and the C,-methylene unit of the hexanoic chain, whereas the latter ion results from the concomitant elimination of the 'COOTMS group and one hydrogen radical from the [M - 151' ion. These fragmentation paths are supported by the corresponding mass shifts observed in the mass spectra of the d,-TMS derivatives.

Although no analytical evidence was obtained from GC/MS analysis of the bacterial extracts to ascertain its route of formation, it is likely from structural analysis that compound 9 originates from the acidic metabolites 10, 11 and/or 12. The absence of hydroxy and keto groups at C-2 and C-6 respectively in compound 9 can be rationalized on the basis of enzymatic or non- enzymatic dehydration reactions of the intermediary a- hydroxy acids, followed by enzymatic reduction of the resulting double bonds and 6-keto function. However, when incubations of the diunsaturated and saturated acids 10, 11 and 12 were carried out in the presence of strain B-206, 4-chlorocinnamic acid (2) was the major metabolite with some 4-CBA, and no trace of com- pound 9 was detected by GC/MS. These results indicate that compound 9, which is a minor metabolite of 4-CB biodegradation in strain B-206, results most probably from enzymatic side reactions which are not activated when the acids 10, 11 and 12 are used as substrates instead of 4-CB. As shown in Schemes 3 and 4, the major pathway of 4-CB degradation in strain B-206 involves the formation of the catechol 8a and its sub- sequent oxidation to the unsaturated acid 10 which upon further oxidation give 4-CBA. About 50% of the initial amount of 4-CB was transformed into 4-CBA after 6 days incubation. It is of interest to note that Catelani and Columbi" reported that biphenyl was transformed in bacteria into 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid, which was further oxidized to yield benzoic acid and 2-hydroxypenta-2,4-dienoic acid. On the basis of the data presented above and pre- viously reported in the l i t e r a t ~ r e , " - ~ ~ * ~ ~ ~ ~ ~ ~ ~ it is likely that 4-CB was degraded according to a similar pathway into 4-CBA by strain B-206, where compounds 7a, 7b, 8a, 8b and 10 are the major metabolic interme- diates.

Thus, compounds 9, 11 and 12 can be considered as by-products of 4-CB metabolism in strain B-206, since no metabolic correlation was established between the latter acids and 4-CBA, the end-product of 4-CB meta- bolic biodegradation.

41' 2 y - c H 2 - c ~ -CH,-CH,-COOTMS

+'*SiMe,

m/z 371 m/z 386[dg-TMS]

-CH COOTMS 4 2 \I:iooTMs

Scheme 7. Formation of ions of m/z 239 and 253 in the mass spectrum of compound 9 TMS derivative.

46 R. M A W ? ET AL.

Finally, three minor metabolites identified as 4- chlorobenzoyl butanoic acid (6) (M", 298; 307, d,-TMS), 4-chlorobenzoyl propionic acid (4) (M +', 284; 293, d,-TMS) and cinnamic acid (2) (M", 254; 263, d,-TMS) were identified in the acidic bacterial extracts (Fig. 7) by comparison with authentic reference com- pounds. Incubation of compounds 4 and 6 with strain B-206 gave 4-chlorocinnamic acid (2) and trace amounts of 4-CBA, whereas compound 2 was recovered unchanged when incubated for 5 days. These data indi- cate that the acids 2,4 and 6 are, as proposed above for compounds 9, 11 and 12 (Scheme 4), by-products of 4-CB metabolism in strain B-206 and do not pertain to the major degradation pathway leading to 4-CBA. Although 3,4-dihydroxy-4'-chlorobiphenyl (8b) was not detected in the bacterial extracts, the formation of the substituted butanoic and pentanoic acids 4 and 6 provide additional evidence for 3,4-dehydroxylation of 4-CB by strain B-206. Indeed, it is likely that com- pounds 4 and 6 originate from enzymatic ortho and meta cleavage of 3,4-dihydroxy-4-chlorobiphenyl according to metabolic routes we have postulated in a previous study.' Similar pathways have been proposed by other authors to account for the formation of chlo- rinated cinnamic, phenylacetic and benzoyl propionic acids formed by the bacterial degradation of several PCB mixtures.*8*20.21

CONCLUSION

The work described above was undertaken as part of a research programme directed toward the study of PCB bacterial metabolism and the development of bacterial strains with the capacity to degrade these ubiquitous pollutants. As one can be seen from Schemes 3 and 4, and on the basis of the GCIMS data presented above, we have shown that the major route of 4-CB degrada- tion in strain B-206 involves 2,3- and 3,Cdihydroxyla- tions by dioxygenases to yield the corresponding 2,3- and 3,4-dihydrodiol intermediates. It is of interest to note that several authors have postulated the formation of 3,4-dihydrodiol intermediates from the bacterial metabolism of PCB congeners in various strain^^^-'^*^^

to account for the formation of specific metabolites. To our knowledge, this is the first report where a 3,4-dihy- drodiol metabolite of a PCB congener (4-CB) is isolated after incubation with a bacterial strain and character- ized by GC/MS analysis. The data also indicate that both dihydrodiols are converted enzymatically to the corresponding catechols. Although 3,4-dihydroxy-4'- chlorobiphenyl (8b) was not detected in the bacterial extract, the acidic metabolites 4 and 6 (Scheme 4) which bear a substituted butanoic and pentanoic chain, respectively, are likely to arise from 3,4-ortho and 2,3- meta oxidative cleavage of 8b.25 The data also suggest that the major metabolic pathway whereby 4-CB is degraded to 4-CBA involves 1,Zmeta cleavage of the catechol 8a to yield the unsaturated acid 10, which upon further oxidation gives 4-CBA. The minor acidic metabolites 2, 4, 6, 9, 11 and 12 are most probably by- products of 4-CB metabolism. Their respective meta- bolic origin could not be determined by incubation of the purified metabolites except for 4-chlorocinnamic acid, which was formed upon incubation of compounds 4,6,10,11 and 12 with strain B-206.

The diunsaturated acidic metabolite resulting from this oxidative cleavage is invariably detected as a mixture of three isomeric forms showing identical mass spectra.

However, the precise metabolic cascade through which 4-chlorobenzoic acid (l), the ultimate biodegra- dation product of 4-CB, is formed is still somewhat speculative since incubation of purified intermediate acid 10 with strain B-206 did not yield CCBA but 4- chlorocinnamic acid as the major biodegradation com- pound. These biotransformations are being actively studied in our laboratory. Nevertheless the mass spec- tral data presented herein should serve as useful analyti- cal criteria for the structural characterization of bacterial metabolites of PCB congeners.

Acknowledgements

The financial support of the Natural Sciences and Engineering Research Council of Canada (grants A-1310, G-1062, G-0942) and the Minisdre de 1'Education du Qubbec (grants Eq-2554 and Eq-2532) is gratefully acknowledged.

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