Mechanism of anaerobic ether cleavage : conversion of 2-phenoxyethanol to phenol and acetaldehyde by Acetobacterium sp.

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Mechanism of Anaerobic Ether Cleavage

CONVERSION OF 2-PHENOXYETHANOL TO PHENOL AND ACETALDEHYDE BYACETOBACTERIUMSP.*

Received for publication, November 19, 2001 Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M111059200

Giovanna Speranza‡§, Britta Mueller^¶, Maximilian Orlandi‡, Carlo F. Morelli‡, Paolo Manitto‡, and Bernhard Schink^¶

From the‡Dipartimento di Chimica Organica e Industriale, Universita` degli Studi di Milano, and Centro di Studio per le Sostanze Organiche Naturali, CNR, via Venezian 21, I-20133 Milano, Italy and the¶Fakultaet fuer Biologie, Universitaet Konstanz, Universitaetsstr. 10, D-78457 Konstanz, Germany

2-Phenoxyethanol is converted into phenol and ace- tate by a strictly anaerobic Gram-positive bacterium, Acetobacteriumstrain LuPhet1. Acetate results from ox- idation of acetaldehyde that is the early product of the biodegradation process (Frings, J., and Schink, B. (1994) Arch. Microbiol. 162, 199 –204). Feeding experiments with resting cell suspensions and 2-phenoxyethanol bearing two deuterium atoms at either carbon of the glycolic moiety as substrate demonstrated that the car- bonyl group of the acetate derives from the alcoholic function and the methyl group derives from the adja- cent carbon. A concomitant migration of a deuterium atom from C-1 to C-2 was observed. These findings were confirmed by NMR analysis of the acetate obtained by fermentation of 2-phenoxy-[2-¹³C,1-²H₂]ethanol, 2-phe- noxy-[1-¹³C,1-²H₂]ethanol, and 2-phenoxy-[1,2-¹³C₂,1-

2H₂]ethanol. During the course of the biotransformation process, the molecular integrity of the glycolic unit was completely retained, no loss of the migrating deuterium occurred by exchange with the medium, and the 1,2- deuterium shift was intramolecular. A diol dehydratase- like mechanism could explain the enzymatic cleavage of the ether bond of 2-phenoxyethanol, provided that an intramolecular H/OC₆H₅ exchange is assumed, giving rise to the hemiacetal precursor of acetaldehyde. How- ever, an alternative mechanism is proposed that is sup- ported by the well recognized propensity of ␣-hy- droxyradical and of its conjugate base (ketyl anion) to eliminate a␤-positioned leaving group.

Ether linkages are comparably stable, and their cleavage requires rather rigorous conditions. Such cleavage reactions represent challenges also to microbes and their enzymes, and this difficulty causes the relative stability of many ether compounds in nature (1).

An important group of xenobiotic ether compounds, the lin- ear polyether PEG¹ and its derivatives, is released into the

environment at high quantities, as lubricants, solubility medi- ators, hydrophilic moiety of nonionic surfactants and house- hold detergents, or as a constituent of cosmetics and pharma- ceutical preparations (2). PEGs were found to be degraded by various bacteria, both in the presence and the absence of molecular oxygen (aerobically, Refs. 2– 6; anaerobically, Refs.

7–12). Different reaction mechanisms are involved in PEG degradation, and it is generally accepted that they all involve the formation of a labile intermediary hemiacetal structure (1).

In the presence of oxygen, such a hemiacetal can be formed through a monooxygenase-catalyzed hydroxylation of one of the methylene carbon atoms. In the absence of molecular oxygen, generation of such a hemiacetal can be achieved only with substrates containing a free hydroxyl group adjacent to the ether carbon through a hydroxyl shift reaction. Such hydroxyl shift reactions are catalyzed by diol dehydratase (EC 4.2.1.28) and glycerol dehydratase (EC 4.2.1.30) enzymes, with the substrates EG, 1,2-propanediol, or glycerol. The reaction mechanisms of these enzymes have been studied in great detail (13–

15). They typically depend on adenosylcobalamin as cofactor, which provides a reversible radical source. Based on these well studied model systems, it was assumed that anaerobic PEG degradation to acetaldehyde as the first identifiable intermediate may be adenosylcobalamin-dependent as well and may proceed in a way analogous to diol dehydratase, provided that at least one terminal hydroxyl group is free for the required shift reaction (7, 10, 11, 16, 17).

The anaerobic homoacetogenic bacterium Acetobacterium strain LuPhet 1 can grow with low molecular weight PEGs as the sole source of carbon and energy but can also use EG or 2-phenoxyethanol as the sole substrate; the latter is fermented to phenol plus acetate (12) as schematized in Fig. 1. In cell-free extracts of this strain, two separate enzyme activities were detected, the one reacting with EG and the other one reacting with phenoxyethanol. Both reactions yield acetaldehyde as the first product. The authors found that the EG-degrading activity was stimulated 3.5-fold by added adenosylcobalamin and was strongly inhibited by cyano- or hydroxocobalamin or by light;

the latter effect could be alleviated by adenosylcobalamin addition (12). With this, the EG-degrading enzyme behaved iden- tically to the known diol dehydratases (18). Cleavage of 2-phenoxyethanol, on the other hand, was influenced neither by various corrinoids, including adenosylcobalamin, nor by light (12), indicating that the two enzymes are definitively different proteins and perhaps operate by different reaction mechanisms.

Since 2-phenoxyethanol is a monosubstituted ethylene glycol, it allows us to study the assumed shift reaction in greater detail because theoretically, either the free hydroxyl group or the phenoxy residue can be shifted to form a hemiacetal as an

* This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft, Bonn in its priority program “Radicals in enzymatic catalysis.” The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dipartimento di Chimica Organica e Industriale, Universita` degli Studi di Milano, via Venezian 21, 20133 Milano, Italy. Tel.: 39-02-5031-4097; Fax: 39-02- 5031-4072; E-mail: giovanna.speranza@unimi.it.

1The abbreviations used are: PEG, polyethylene glycol; EG, ethylene glycol; TLC, thin layer chromatography; FC, flash chromatography; GC, gas chromatography; EIMS, electron impact mass spectrometry; rel.

int., relative intensity.

THEJOURNAL OFBIOLOGICALCHEMISTRY Vol. 277, No. 14, Issue of April 5, pp. 11684 –11690, 2002

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intermediate. We therefore tried to distinguish between those two possible pathways by application of specifically deuterated and/or ¹³C-labeled 2-phenoxyethanol preparations to resting cell suspensions ofAcetobacteriumstrain LuPhet 1 and subsequent analysis of the produced acetate.

EXPERIMENTAL PROCEDURES

General Methods—TLC was performed on Silica Gel F254-precoated aluminum sheets (0.2-mm layer, Merck, Darmstadt, Germany); compo- nents were detected by spraying a ceric sulfate ammonium molybdate solution followed by heating to⬃150° C. Silica gel (Merck, 40 – 63␮m) was used for FC. GC analyses were carried out on a DANI 3800 gas chromatograph (DANI, Monza, Italy) using a homemade glass column (2 m ⫻ 2 mm inner diameter) packed with 20% Carbowax 20M on Chromosorb W (60 – 80 mesh). GC parameters were as follows: injector, 220 °C; detector (flame ionization detection), 220 °C; carrier, N2(30 ml/min); oven, from 60 to 200 °C at 10 °C/min.¹H and¹³C NMR spectra were acquired at 400.132 and 100.613 MHz on a Bruker AVANCE 400 Spectrometer using an Xwin-nmr software package and at 200.133 and 50.330 MHz on a Bruker AC 200 (Bruker, Karlsruhe, Germany) equipped with an ASPECT 2000 data system. Chemical shifts (␦) are given in parts per million and were referenced to the signals of CDCl3

(␦H7.25 and␦C77.00 ppm) or to 3-(trimethylsilyl)propionic-2,2,3,3-d4

acid sodium salt (␦Me 0 ppm) in the case of D2O/NaOD (pH ⬎10) solutions.¹³C NMR signal multiplicities were based on attached proton test spectra.¹³C NMR spectra for quantitative analyses were obtained by the inverse gated decoupling pulse sequence and a relaxation delay of 300 s (19). EIMS spectra were run on a VG 7070 EQ mass spectrometer (VG Instruments, Manchester, UK) operating at 70 eV. All re- agents were of commercial quality or purified prior to use by standard methods. Ethyl bromo-[2-¹³C]acetate, bromo-[1-¹³C]acetate, and bromo- [1,2-¹³C]acetate were from Aldrich.

Medium and Growth Conditions—Acetobacteriumstrain LuPhet 1 (DSM 9077) was grown at 28 °C in the dark in bicarbonate-buffered (30 mM, pH 7.2), sulfide-reduced (1 mM) freshwater mineral medium (20) with 10 mM2-phenoxyethanol as sole organic carbon substrate under a N2/CO2 atmosphere (80:20 v/v) as described previously (12). 2-Phe- noxyethanol was added from anoxic filter-sterilized stock solutions.

Besides other vitamins, the medium contained about 40 nMcyanocobal- amin. The addition of a few crystals of dithionite shortened the lag phases. Cells were grown as batch cultures of 0.5- or 1-liter volume in infusion bottles sealed with butyl rubber septa. Growth was followed by measuring turbidity at 578 nm.

Cell Suspension Experiments with Labeled 2-Phenoxyethanol—Cell suspensions were prepared under strictly anoxic conditions in an anoxic chamber (Coy Laboratory Products, Ann Arbor, MI) with an atmosphere of 5% H2in N2. Bacteria were harvested in the late exponential growth phase (A578⫽0.1) by centrifugation at 11,000⫻gand 4 °C for 30 min. Polypropylene centrifuge beakers were preincubated in the chamber for 2–3 days. Cells were washed once with degassed potassium phosphate buffer (50 mM, pH 7.0) prereduced with 2.5 mMtitanium(I- II)citrate and then resuspended in freshwater mineral medium without substrate (bicarbonate-buffered, 30 mM, pH 7.2, and sulfide-reduced, 1 mM) and transferred into a serum bottle sealed with a butyl rubber stopper. The headspace in the bottle was exchanged to N2/CO2(80:20 v/v), and the cell suspension was incubated at 28 °C under protection from light. The reaction was started by the addition of labeled 2-phenoxyethanol to about 10 mMconcentration. Aliquots (50␮l) were taken at regular intervals with a gas-tight syringe and injected into 200␮l of H3PO4 (100 mM) to stop all enzymatic reactions. 2-Phenoxyethanol, phenol, and acetate were analyzed with a high performance liquid chromatography system (System Gold, Beckman Instruments) equipped with an AQ-ODS column (4.6 by 250 mm) from YMC Europe

(Schermbeck, Germany) with an eluent composed of ammonium phosphate buffer (100 mM, pH 2.6) and methanol. The three compounds were measured simultaneously using a gradient from 5% methanol increasing to 60% methanol and detection at a 206-nm wavelength.

Concentrations were calculated via external standards. The protein content in the cell suspension varied between 0.09 and 0.4 mg/ml. The reaction was stopped after substrate depletion or after a maximum of 28 h by centrifugation at 11,000 ⫻ g for 30 min and at 4 °C. The supernatant was filtered through a cellulose acetate membrane filter with a pore size of 0.2␮m and stored at 4 °C. From the supernatant the acetate was isolated by the procedure described below.

Isolation of Acetate from the Reaction Mixture—The neutral or slightly alkaline aqueous phase was extracted three times with chloro- form to reduce the phenol and 2-phenoxyethanol content before acidi- fication to pH 1–2 by the addition of concentrated HCl. Then some NaCl was added to the aqueous phase, and the acetic acid was extracted with diethyl ether at least twice with a 5:1 ether-to-water volume ratio. The ether phase was concentrated to few millilitersin vacuo, and the acetic acid was dissociated by the addition of a sufficient amount of sodium hydroxide (2 M) and freeze-dried. Sodium acetate showed chemical shifts in the range␦H1.88 –2.03 (literature 1.90),␦C23.8 –26.3 (literature 23.97), and␦C182.0 –184.4 (literature 182.02) (21) for the methyl and the carbonyl group, respectively.

2-Phenoxy-[1-²H2]ethanol (3)—This substrate was prepared according to Ref. 22 with modifications as follows. A solution of sodium phenoxide trihydrate (340 mg, 2 mmol) and ethyl bromoacetate (1) (300 mg, 1.8 mmol) in ethanol (5 ml) was refluxed under N2, monitoring the reaction progress by TLC (petroleum ether/diethyl ether, 8:2) and GC.

After 4 h, the reaction mixture was diluted with water (10 ml), acidified with 2NHCl, and extracted with diethyl ether. The organic phase was washed with saturated NaHCO3, washed with water, and dried over Na2SO4. Solvent removal under reduced pressure followed by FC of the residue (eluent as above) afforded ethyl phenoxyacetate (2) (230 mg, 71% yield); pure by TLC (Rf0.61) and GC (tR6.8 min),¹H NMR and EIMS as in Ref. 23;¹³C NMR as in Ref. 24. Compound2(200 mg, 1.1 mmol) in dry diethyl ether (2 ml) was added dropwise to a cold (0 °C) suspension of LiAlD4(92.4 mg, 2.2 mmol) in dry diethyl ether (4 ml), and the reaction mixture was refluxed with stirring under N2for 5 h (GC control). After cooling to room temperature, a saturated solution of Na2SO4was carefully added. The white salts were removed by filtration and then washed with diethyl ether. The filtrate was washed with water, dried (Na2SO4), and evaporated under reduced pressure to give the title compound3(142 mg, 92% yield) pure by GC (tR8.5 min);¹H NMR and EIMS as in Ref. 22;¹³C NMR (CDCl3, 50 MHz)␦60.71 (CD2, quintet,¹JCD⫽21.4 Hz), 68.96 (CH2), 114.54 (CH), 121.07 (CH), 129.45 (CH), 158.57 (C).

2-Phenoxy-[2-²H2]ethanol (7)—Benzyloxyacetyl chloride (4.7 g, 25.5 mmol) was added via a syringe over 15 min to an ice-cooled solution of pyridine (5 ml) and ethyl alcohol (4 ml) in dry dichloromethane (15 ml) under N2with stirring. The reaction mixture was allowed to warm to room temperature, and stirring was continued for 30 min followed by quenching with 1NHCl (20 ml). The two phases were separated, and the organic one was washed with water (2⫻20 ml), dried over Na2SO4, and concentrated under reduced pressure to give a viscous oil. After purification by FC (petroleum ether/ethyl acetate, 5:2), ethyl benzyloxy- acetate (4) (4.7 g, 95% yield) was obtained, pure by TLC (Rf0.46, eluent as above),¹H, and¹³C NMR (25). To an ice-cooled solution of the ester 4(4.7 g, 24.2 mmol) in dry diethyl ether (40 ml) was added LiAlD4(1.2 g, 47.6 mmol) in several portions. After further addition of diethyl ether (10 ml), the reaction mixture was refluxed for 3 h. Workup as described above for compound3afforded 2-benzyloxy-[1,1-²H2]ethanol (5)(3.3 g, 89% yield), pure by TLC (Rf0.15, eluent as above) and GC (tR8.7 min), which was used for the next step without further purification;¹H NMR and EIMS as in Ref. 26; ¹³C NMR (CDCl3, 50 MHz)␦61.11 (CD2, quintet,¹JCD⫽21.9 Hz), 71.35 (CH2), 73.30 (CH2), 127.81 (CH), 128.47 (CH), 138.00 (C).

A stirred solution of PPh3(2.0 g, 7.8 mmol) and diisopropyl azodicarboxylate (1.5 ml, 7.8 mmol) in tetrahydrofuran (80 ml) at 0 °C was treated, sequentially, with a solution of freshly distilled phenol (1.1 g, 11.7 mmol) in tetrahydrofuran (5 ml) and then with a solution of 2-benzyloxy-[1,1-²H2]ethanol (5) (1.0 g, 6.5 mmol) over a period of 15 min. The reaction mixture was allowed to warm to room temperature, stirred for an additional 1 h (TLC control), and quenched by the addition of water (5 ml) and a few drops of concentrated HCl. The solvent was removed under reduced pressure, and the residue was taken up with diethyl ether (60 ml). Insoluble materials were removed by filtration, and the filtrate was washed with 2NNaOH and with water, dried (Na2SO4), and concentrated to approximately a half-volume under re- FIG. 1.Pathway for anaerobic degradation of phenoxyethanol

by strain LuPhet 1. The acetaldehyde formed in phenoxyethanol cleavage is oxidized to acetate by an acetaldehyde:acceptor oxidoreduc- tase that forms acetyl coenzyme A. The reducing equivalents are used to reduce carbon dioxide to acetate through the carbon monoxide dehydrogenase pathway (see Ref. 12).

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duced pressure. After further filtration of insoluble materials, the solvent was removed under reduced pressure, and the residue was purified by FC (petroleum ether/ethyl acetate, 9:1) to give pure6(1.1 g, 73%

yield);¹H NMR (CDCl3, 200 MHz)␦3.84 (s, 2H), 4.65 (s, 2H), 6.94 – 6.99 (m, 3H), 7.27–7.41 (m, 7H);¹³C NMR (CDCl3, 50 MHz)␦67.02 (CD2, quintet,¹JCD⫽21.9 Hz) 68.48 (CH2), 73.42 (CH2), 114.72 (CH), 120.90 (CH), 127.76 (CH), 128.42 (CH), 129.45 (CH), 138.18 (C), 158.86 (C).

2-Benzyloxy-1-phenoxy-[1-²H2]ethane (6) (900 mg, 3.9 mmol) was dis- solved in MeOH (90 ml) and hydrogenated in the presence of 10%

palladium on activated carbon (570 mg) under 1 atm of hydrogen at room temperature for 1 h (TLC control). Filtration of the catalyst and removal of the solvent under reduced pressure gave the desired product (7) in quantitative yield (540 mg) as a colorless oil:¹H NMR (CDCl3, 200 MHz)␦2.10 (s, 1H, OH), 3.94 (s, 2H), 6.91–7.01 (m, 3H), 7.26 –7.33 (m, 2H);¹³C NMR (CDCl3, 50 MHz)␦61.17 (CH2), 68.34 (CD2, quintet,¹JCD

⫽21.9 Hz); EIMSm/z(rel. int.) 140 (M^⫹, 25), 109 (10), 94 (100), 77 (35).

(¹³C,²H)-Labeled 2-Phenoxyethanols (8 –10)—These substances were obtained using differently¹³C-labeled ethyl bromoacetate and LiAlD4

according to the procedure described above for 2-phenoxy-[1-

2H2]ethanol (3). Ethyl phenoxy-[2-¹³C]acetate:¹H NMR (CDCl3, 200 MHz)␦1.29 (t, 3H,J⫽7.1 Hz), 4.27 (q, 2H,J⫽7.1 Hz), 4.61 (d, 2H,

1JCH⫽146.0 Hz), 6.80 –7.02 (m, 3H), 7.19 –7.34 (m, 2H); EIMSm/z(rel.

int.) 181 (M^⫹, 90), 108 (100), 94 (25), 77 (80); after dilution with unlabeled ethyl phenoxyacetate (2), it gave compound 8: ¹H NMR (CDCl3, 200 MHz)␦1.97 (s, 1H, OH), 4.08 (s, 1.84H and d, 0.16H,¹JCH

⫽143.3 Hz), 6.91–7.01 (m, 3H), 7.26 –7.33 (m, 2H);¹³C NMR (CDCl3, 50 MHz)␦68.98 (¹³CH2). Ethyl phenoxy-[1-¹³C]acetate:¹H NMR (CDCl3, 200 MHz)␦4.27 (dq, 2H,³JCH⫽3.1 Hz,J⫽7.1 Hz), 4.61 (d, 2H,²JCH

⫽4.7 Hz); EIMSm/z(rel. int.) 181 (M^⫹, 95), 107 (100), 94 (30), 77 (80);

it gave9:¹H NMR (CDCl3, 200 MHz)␦4.08 (s, 2H);¹³C NMR (CDCl3, 50 MHz)␦60.84 (¹³CD2, quintet,¹JCD⫽21.4 Hz); EIMSm/z(rel. int.) 141 (M^⫹, 20), 107 (10), 94 (100), 77 (35). Ethyl phenoxy-[1,2-

13C2]acetate:¹H NMR (CDCl3, 200 MHz)␦4.27 (dq, 2H,³JCH⫽3.1 Hz, J⫽7.1 Hz), 4.61 (dd, 2H,¹JCH⫽146.0 Hz,²JCH⫽4.7 Hz);¹³C NMR (CDCl3)␦65.48 (¹³CH2, d,¹JCC⫽64.5 Hz), 169.12 (¹³CO, d,¹JCC⫽64.5 Hz); EIMSm/z(rel. int.) 182 (M^⫹, 90), 108 (100), 94 (25), 77 (80); it gave 10:¹H NMR (CDCl3)␦4.08 (d, 2H,¹JCH⫽142.8 Hz);¹³C NMR (CDCl3)

␦60.98 (¹³CD2, doublet of quintets,¹JCC⫽39.3 Hz,¹JCD⫽21.4 Hz), 69.08 (¹³CH2, d,¹JCC⫽39.3 Hz); EIMSm/z(rel. int.) 142 (M^⫹, 20), 108 (5), 94 (100), 77 (35).

RESULTS

2-Phenoxyethanol dideuterated at carbon-1 (3, D₂-molecules⬎98%) was prepared by LiAlD₄reduction of ethyl 2-phenoxyacetate (2) obtained, in turn, by the reaction of sodium phenoxide with ethyl 2-bromoacetate (1) (22) (Fig. 2A). After

the complete fermentation of 3 by Acetobacterium under a N₂/CO₂atmosphere, sodium acetate was isolated from the cul- ture supernatant and examined by¹H and¹³C NMR spectroscopy. It is understood that throughout this study, spectra of sodium acetate (proton-decoupled in the case of¹³C) were re- corded using NaOD/D₂O at pH⬎ 10. The methyl regions of these spectra exhibited peaks assignable to a mixture of mono- and non-deuterated acetate molecules only (Fig. 3,AandB).

Monodeuterated molecules are revealed by the typical patterns of ¹H and ¹³C NMR signals due to the CH₂D and ¹³CH₂D groups. In both cases, this pattern consists of a 1:1:1 triplet (27) (²J_HD⫽2.09 Hz,J_CD⫽19.5 Hz) (28, 29), which is upfield with respect to the non-deuterated methyl group (²⌬H(D) ⫽13.5 ppb,⌬C(D)⫽0.254 ppm) (29, 30). The presence of non-deuterated molecules besides the monodeuterated ones in the fermentation acetate (⬃35% as calculated from the integrated peak areas in the¹H NMR spectrum, taking into account the number of protons of the two species) can be explained by considering additional acetate synthesis from CO₂by this acetogenic bacterium (12) (Fig. 1). In addition, a partial loss of both deuterium atoms during the conversion of 2-phenoxyethanol into phenol and acetate could not be excluded.

When Acetobacterium cells were fed with 2-phenoxy-[2-

2H₂]ethanol (7) prepared as shown in Fig. 2B, the resulting acetate was found to be a mixture of dideuterated and non- deuterated molecules in the ratio⬃2.5:1. In fact, in the¹H and

13C NMR spectra of this acetate, an upfield quintet (1:2:3:2:1) (27) was present beside the singlets due to the non-deuterated methyl group (²⌬H(D₂)⫽27.0 ppb,⌬C(D₂)⫽0.467 ppm) (29, 30), thus indicating the occurrence of CHD₂ and ¹³CHD₂ groups (Fig. 3,CandD). The complete absence of CHD₂-CO₂^⫺ and CH₂D-CO₂^⫺species in the product from the former and the latter experiment, respectively, clearly resulted from a compar- ison of the corresponding NMR spectra. The results of the experiments carried out with 2-phenoxyethanol bearing the dideuterated methylene group at either position of the glycol unit were in agreement with each other and consistent with the conversion of carbon-1 into the carboxylic group of the acetate and of carbon-2 into the methyl group. The most striking fea- ture of this biotransformation appeared to be the shift of a deuterium (hydrogen) atom from carbon-1 to carbon-2 (Reac- tion 1).

C6H5OH

 ‘

C6H5O* CH2⫺y

CD2OHO¡

1,2-D shift

CH* 2D⫺y

CDOO[O]¡* CH2D⫺y

CO2⫺

REACTION1

To gain further insight into the process schematized in Re- action 1, samples of 2-phenoxyethanol enriched with¹³C at 1- and/or 2-position and dideuterated at the alcoholic function,i.e.

8, 9, and 10 (Fig. 4), were prepared from the proper ethyl [¹³C]bromoacetate. The quantitative determination of differently labeled species (isotopomers) in the acetate recovered from feeding experiments performed with these samples was based on peak area measurements in¹H and¹³C NMR spectra, provided that the latter were obtained by the inverse gated decoupling method (19). The identification of signals due to isotopomeric molecules was made possible by exploiting deuterium effects on the shielding of¹H and¹³C nuclei as well as spin-spin coupling constants.

After fermentation of sample8,the¹H NMR spectrum of the resulting acetate showed signals of CH₂D (triplet) and CH₃at a ratio from which ⬃45% dilution of the biotransformation product withde novosynthesized acetate could be calculated (neglecting satellite peaks due to¹³CH₂D and¹³CH₃groups). A FIG. 2.Synthesis of labeled substrates.The following abbrevia-

tions are used: Ph, C6H5; EtOH, ethanol; Et2O, diethyl ether; Bn, CH2C6H5;DIAD, diisopropyl azodicarboxylate;THF, tetrahydrofuran;

Pd-C, palladium on activated carbon. For details on the syntheses, see

“Experimental Procedures.”

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complete retention of the migrating deuterium atom (within the limits of the experimental error) was indicated by the

13CH₃/CH₃ peak area ratio approximating the value of ¹³C natural abundance (⬃1.1%). In accordance with this assump- tion, the peak intensities measured in the¹³C NMR spectrum appeared in the expected proportions,i.e.⬃1:16:3 for the¹³CH₃ group (singlet at␦ 26.27, acetate coming from the acetogenic activity of the microorganism), for the¹³CH₂D group (triplet

upfield shifted, acetate coming from the 2-phenoxyethanol sup- plied), and for the¹³CO₂^⫺(singlet at␦184.39, corresponding to the¹³C natural abundance level of the whole acetate recovered from the fermentation experiment).

Only the peak due to the [¹³C]carboxylate group was detect- able in the¹³C NMR spectrum of the acetate arising from the bioconversion of9. The¹H NMR spectrum displayed singlet at

␦ 2.031 and a 1:1:1:1:1:1 system centered at␦2.017, really a doublet (²J_HC⫽5.9 Hz) (31) of triplet (²J_HD) in agreement with the presence of two species only, i.e. CH₃-CO₂^⫺ and CH₂D-

13CO₂^⫺, in the ratio of ⬃1:2. The absence of the isotopomer CH₂D-CO₂^⫺allows the exclusion of an exchange with the medium of the carbonyl group (at the level of acetyl-CoA) (Fig. 1).

Such an exchange has been reported to occur by the action of carbon monoxide dehydrogenase (32), an enzyme that is present in our strain ofAcetobacterium(12).

Assuming the participation of the enzyme/coenzyme system as a hydrogen carrier in the hydrogen 1,2-shift during the biodegradation of 2-phenoxyethanol, two possibilities could be envisaged: (i) the hydrogen (deuterium) atom is abstracted from a substrate molecule, temporarily retained by the enzyme, FIG. 3. ¹H (400 MHz) and ¹³C (100

MHz) NMR spectra of sodium acetate (methyl group resonances only) com- ing from fermentation of 2-phenoxy- [1-²H2]ethanol (3) (A, B) and 2-phe- noxy-[2-²H2]ethanol (7) (C, D). For values of coupling constants and isotope shifts, see text.

FIG. 4.¹³C,²H-labeled substrates used in feeding experiments.

Compounds8,9, and10were prepared according toScheme Aof Fig. 2 starting from commercially available Br¹³CH2COOCH2CH3, BrCH2

13COOCH2CH3, and Br¹³CH2

13COOCH2CH3, respectively.

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and then transferred to another molecule (intermolecular transfer) or (ii) the migrating hydrogen (deuterium) is returned to the same glycolic unit from which it had been abstracted (enzyme-mediated intramolecular transfer). To estimate the relative extent of the two events, compound10was adminis- tered to a cell suspension ofAcetobacteriumafter dilution (18 to 100) with unlabeled 2-phenoxyethanol. When the acetate isolated at the end of this fermentation was examined by¹H NMR (Fig. 5A), no signals assignable to the CH₂D group were ob-

served,i.e.no signals of a triplet 13.5 ppb upfield shifted from the singlet due to the CH₃ group (Fig. 3A). This result was consistent with a complete intramolecularity of the C-1 hydrogen migration. In addition, well resolved systems of satellite peaks were present in the proton NMR spectrum (Fig. 5A) due to isotopomers containing¹³CH₃and¹³CH₂D groups. The mul- tiplicity of the system corresponding to the [¹³C,D]methyl group, i.e. a doublet of 1:1:1:1:1:1 sextets (doublet centered upfield with respect to the CH₃singlet in agreement with the FIG. 5.¹H (400 MHz) (A) and¹³C (50 MHz) NMR spectra (BandC) of acetate isolated from fermentation of a 1:5.5 mixture of 2-phenoxy-[1,2-¹³C2, 1-²H2]ethanol (intramolecular isotopic substitution>98%), and 2-phenoxyethanol having natural nuclidic composition.Signal assignments are as follows:a,¹³CH2D-¹³CO2⫺;b, CH3-¹³CO2⫺;c,¹³CH3CO2⫺;d,¹³CH2D-CO2⫺;e,¹³CH3-¹³CO2⫺.

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expected deuterium isotope shift), was indicative of a strong prevalence of ¹³CH₂D-¹³CO₂^⫺ species among the [¹³C]methyl isotopomeric mixture (J_HC⫽126.7 Hz) (31). The presence of a very minor concentration of¹³CH₃CO₂^⫺isotopomer was recog- nizable by the slightly higher intensity of the downfield peak of each sextet and could be explained in terms of natural ¹³C abundance in the acetate molecule accompanying the doubly

13C-labeled ones.

These findings were corroborated by the following consider- ations: (i) by inspection of methyl and carboxyl regions of the

13C NMR spectrum (Fig. 5,BandC) the composition of the¹³C isotopomeric mixture was estimated to be: ¹³CH₂D-¹³CO₂^⫺ (a)⫽86% (dt at␦26.05 and d at␦184.40;J_CC⫽52 Hz,J_CD⫽ 19.5 Hz) (28, 31); CH₃-¹³CO₂^⫺ (b) ⫽ 6% (s at ␦ 184.42);

13CH₃CO₂^⫺(c)⫽6% (s at␦26.29);¹³CH₂D-CO₂^⫺(d)ⱕ1% (t at

␦ 26.05,J_CD);¹³CH₃-¹³CO₂^⫺(e)ⱕ1% (d at␦26.29 and d at␦ 184.40,J_CC); (ii) the ratio of acetate resulting from acetogenic activity to that produced by transformation of 2-phenoxyethanol was found to be 1:2.6. This value was calculated from the ratio between CH₃-CO₂^⫺molecules (measured as the area of the singlet at␦H1.90) and¹³CH₂D-¹³CO₂molecules (measured as the total area of the satellite signals, decreased by 1.1% of the CH₃area and then corrected for the number of hydrogen atoms in the monodeuterated methyl group), taking into account the concentration (18%) of the labeled substrate in the sample fermented; (iii) the percentage of isotopomersbandcin the¹³C isotopomeric mixture was found to be very close to the expected one (⫾5%) for¹³C-labeled species present at the natural abundance level in the portion of acetate (87% of the total) arising in part (28%) from de novo synthesis and in part (59%) from 2-phenoxyethanol used to dilute the doubly labeled substrate.

As regards the isotopomersdande, which are present in trace amount in the acetate examined, their formation might depend on hydrogen and CO exchange reactions (32) occurring to a very small extent. Thus, the conversion of 2-phenoxyethanol into acetate appears to be an essentially straightforward process, as shown in Reaction 1, involving an intramolecular hydrogen migration in the first step.

DISCUSSION

In the light of a previous report (12) and in light of the results obtained by feeding experiments performed using¹H- and¹³C- labeled substrates and resting cell suspensions ofAcetobacte- riumstrain LuPhet1, the conversion of 2-phenoxyethanol into acetate and phenol can be summarized as follows. Acetate originates from the glycolic moiety of 2-phenoxyethanol through elimination of phenol with formation of acetaldehyde, which is then oxidized in subsequent steps with retention of its molecular integrity (Fig. 1). In the first reaction, the alcoholic function of the substrate becomes a formyl group, whereas the adjacent methylene group is transformed into a methyl group with concomitant 1,2-hydrogen shift (Reaction 1). These fea- tures are strongly reminiscent of the dioldehydratase-catalyzed reactions for which a generally accepted mechanism is schematized in Fig. 6A for 1,2-ethanediol (11,R⫽ H) (18, 33). The whole process encompasses a double H/OH interchange giving rise to the gem-diol (14, R ⫽ H) (15, 34, 35) that rapidly collapses to the aldehyde15. Its radical nature has largely been proven (33, 36, 37) and appears to be consistent with the transfer of a hydrogen atom from the C-1 of the substrate to a transient radical (X䡠) and then back to C-2 of the product- related radical (13,R⫽H), generated in turn by a hydroxyl 1,2-shift.

If an analogous rearrangement occurs in the anaerobic degradation of 2-phenoxyethanol (11,R⫽C₆H₅) with formation of the labile hemiacetal (14, R ⫽ C₆H₅), the migration of the phenoxyl group should be assumed given the metabolic corre-

lation between each carbon atom of the glycolic unit of 2-phenoxyethanol and those of the acetate molecule (Reaction 1).

Thus, the opposite pathway, i.e. the 1,2-hydroxyl shift suggested previously (11, 12), has to be ruled out.

Considering that no evidence has been given so far for the formation of the hemiacetal (14, R ⫽C₆H₅), an alternative mechanism can be envisaged with regard to the subsequent transformation of the radical intermediate (12,R⫽C₆H₅) (Fig.

6A). This mechanism (Fig. 6B), based on the intermediacy of the resonance stabilized (␣-carbonyl7enoxy) radical18(33), is supported by the propensity of ketyls (radical anions) (e.g.

17) to eliminate adjacent leaving groups as a result of their electron-rich character (38, 39). The cleavage of the␤-C,O-bond can also be facilitated by stereoelectronic effects in the appro- priate conformation of the radical anion 17 (40). It is well known that␣-hydroxy radicals are up to 10⁵times more acidic than the corresponding alcohols (CH₂OH-䡠CHOH has pK_aval- ues of ⬃10 –12) (41). In addition, a base-promoted hydrogen abstraction as schematized in formula16is coherent with the marked lowering of gas-phase C–H bond dissociation energy observed when going from 1-alkanols (e.g.94⫾2 kcal mol^⫺1for H-CH₂OH) (42) to alcoholate ions (e.g. 85 kcal mol^⫺¹ for H-CH₂O^⫺) (43).␣-Oxo radicals have been proposed as interme- diates in a number of enzymatic reactions (36, 38, 39, 43, 44).

We have found that in the biotransformation of 2-phenoxyethanol, the exchange of the migrating hydrogen atom with the medium occurs only to a negligible extent, if at all, and that its 1,2-shift is intramolecular (even if enzyme-mediated).

FIG. 6.Hypothetical reaction mechanisms for anaerobic glycol ether cleavage. A, commonly accepted reaction mechanism of diol dehydratases (R⫽H);X䡠denoting 5⬘-deoxyadenosyl radical or a protein-based radical.B, putative mechanism of the enzyme-catalyzed C-O cleavage of phenoxyethanol byAcetobacteriumsp.;X䡠denotes a protein- based radical.C, alternative pathway for the conversion of␣-oxo radical 18into acetaldehyde;NuH, nucleophile (e.g.H2O).

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The fact that the hydrogen atom abstracted from the C-1 position of the substrate is returned quantitatively to the adjacent position of the same molecule requires that the hydrogen carrier be monoprotic (XHin Fig. 6B). It can be noted that such a facet of the phenoxyethanol acetaldehyde lyase recalls the reaction mechanism of adenosylcobalamin-dependent ribonucle- otide reductase ofLactobacillus leichmannii, which involves a protein-based cysteinyl radical as a catalytically competent intermediate (43). Although the ␣-oxo radicals appear to be thermodynamically capable of hydrogen abstraction from a thiol group (XH⫽Enz-SH in Fig. 6B), given that gas-phase bond dissociation energies of H-SR compounds are in the range 88 –92 kcal mol^⫺1 (43) and bond dissociation energy of H-CH₂COCH₃was estimated at⬃91 kcal mol^⫺1(45), a tempo- rary addition of a nucleophile to the carbonyl group of the radical18might occur (Fig. 6C). This further step would re- move the resonance stabilization in 18(⬃8 kcal mol^⫺1) (see Table IV, footnote k in Ref. 45), thus facilitating the formation of the C–H bond by the intermediate19to give the labile diol 20(bond dissociation energy for H-CH₂R⬃98 kcal mol^⫺1) (42).

A similar addition (with NuH⫽H₂O) has been suggested in the case of the diol dehydratase reaction mechanism (18, 36, 37, 39). It remains to be elucidated whether this reaction mechanism also underlies anaerobic cleavage of PEG and its derivatives.

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