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J. Biol. Chem., Vol. 277, Issue 14, 11684-11690, April 5, 2002

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Mechanism of Anaerobic Ether Cleavage

CONVERSION OF 2-PHENOXYETHANOL TO PHENOL AND ACETALDEHYDE BY ACETOBACTERIUM SP.^*

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

From the  Dipartimento di Chimica Organica e Industriale, Università 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

Received for publication, November 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

2-Phenoxyethanol is converted into phenol and acetate by a strictly anaerobic Gram-positive bacterium, Acetobacterium strain LuPhet1. Acetate results from oxidation 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 carbonyl group of the acetate derives from the alcoholic function and the methyl group derives from the adjacent 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-phenoxy-[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. However, an alternative mechanism is proposed that is supported by the well recognized propensity of -hydroxyradical and of its conjugate base (ketyl anion) to eliminate a -positioned leaving group.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 linear polyether PEG¹ and its derivatives, is released into the environment at high quantities, as lubricants, solubility mediators, hydrophilic moiety of nonionic surfactants and household detergents, or as a constituent of cosmetics and pharmaceutical 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 identically 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.

View larger version (8K): [in this window] [in a new window]   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 oxidoreductase 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).

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 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 of Acetobacterium strain LuPhet 1 and subsequent analysis of the produced acetate.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

General Methods-- TLC was performed on Silica Gel F₂₅₄-precoated aluminum sheets (0.2-mm layer, Merck, Darmstadt, Germany); components were detected by spraying a ceric sulfate ammonium molybdate solution followed by heating to ~150° C. Silica gel (Merck, 40-63 µm) 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 and ¹³C 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 (_H 7.25 and _C 77.00 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 reagents 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-- Acetobacterium strain 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 mM 2-phenoxyethanol as sole organic carbon substrate under a N₂/CO₂ atmosphere (80:20 v/v) as described previously (12). 2-Phenoxyethanol was added from anoxic filter-sterilized stock solutions. Besides other vitamins, the medium contained about 40 nM cyanocobalamin. 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% H₂ in N₂. Bacteria were harvested in the late exponential growth phase (A₅₇₈ = 0.1) by centrifugation at 11,000 × g and 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 mM titanium(III)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 mM concentration. Aliquots (50 µl) were taken at regular intervals with a gas-tight syringe and injected into 200 µl 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 µm 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 chloroform to reduce the phenol and 2-phenoxyethanol content before acidification 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 milliliters in 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 _H 1.88-2.03 (literature 1.90), _C 23.8-26.3 (literature 23.97), and _C 182.0-184.4 (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 2 N HCl, 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 (R_f 0.61) and GC (t_R 6.8 min), ¹H NMR and EIMS as in Ref. 23; ¹³C NMR as in Ref. 24. Compound 2 (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 N₂ for 5 h (GC control). After cooling to room temperature, a saturated solution of Na₂SO₄ was 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 compound 3 (142 mg, 92% yield) pure by GC (t_R 8.5 min); ¹H NMR and EIMS as in Ref. 22; ¹³C NMR (CDCl₃, 50 MHz)  60.71 (CD₂, quintet, ¹J_CD = 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 N₂ with stirring. The reaction mixture was allowed to warm to room temperature, and stirring was continued for 30 min followed by quenching with 1 N HCl (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 benzyloxyacetate (4) (4.7 g, 95% yield) was obtained, pure by TLC (R_f 0.46, eluent as above), ¹H, and ¹³C 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 compound 3 afforded 2-benzyloxy-[1,1-²H₂]ethanol (5) (3.3 g, 89% yield), pure by TLC (R_f 0.15, eluent as above) and GC (t_R 8.7 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, ¹J_CD = 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 2 N NaOH and with water, dried (Na₂SO₄), and concentrated to approximately a half-volume under reduced 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 pure 6 (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, ¹J_CD = 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 dissolved 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, ¹J_CD = 21.9 Hz); EIMS m/z (rel. int.) 140 (M⁺, 25), 109 (10), 94 (100), 77 (35).

(¹³C, ²H)-Labeled 2-Phenoxyethanols (8-10)-- These substances were obtained using differently ¹³C-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, ¹J_CH = 146.0 Hz), 6.80-7.02 (m, 3H), 7.19-7.34 (m, 2H); EIMS m/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, ¹J_CH = 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, ³J_CH = 3.1 Hz, J = 7.1 Hz), 4.61 (d, 2H, ²J_CH = 4.7 Hz); EIMS m/z (rel. int.) 181 (M⁺, 95), 107 (100), 94 (30), 77 (80); it gave 9: ¹H NMR (CDCl₃, 200 MHz)  4.08 (s, 2H); ¹³C NMR (CDCl₃, 50 MHz)  60.84 (¹³CD₂, quintet, ¹J_CD = 21.4 Hz); EIMS m/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, ³J_CH = 3.1 Hz, J = 7.1 Hz), 4.61 (dd, 2H, ¹J_CH = 146.0 Hz, ²J_CH = 4.7 Hz); ¹³C NMR (CDCl₃)  65.48 (¹³CH₂, d, ¹J_CC = 64.5 Hz), 169.12 (¹³CO, d, ¹J_CC = 64.5 Hz); EIMS m/z (rel. int.) 182 (M⁺, 90), 108 (100), 94 (25), 77 (80); it gave 10: ¹H NMR (CDCl₃)  4.08 (d, 2H, ¹J_CH = 142.8 Hz); ¹³C NMR (CDCl₃)  60.98 (¹³CD₂, doublet of quintets, ¹J_CC = 39.3 Hz, ¹J_CD = 21.4 Hz), 69.08 (¹³CH₂, d, ¹J_CC = 39.3 Hz); EIMS m/z (rel. int.) 142 (M⁺, 20), 108 (5), 94 (100), 77 (35).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

2-Phenoxyethanol dideuterated at carbon-1 (3, D₂-molecules > 98%) was prepared by LiAlD₄ reduction 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/CO₂ atmosphere, sodium acetate was isolated from the culture supernatant and examined by ¹H and ¹³C NMR spectroscopy. It is understood that throughout this study, spectra of sodium acetate (proton-decoupled in the case of ¹³C) were recorded 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, A and B). 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 (²H(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 the ¹H NMR spectrum, taking into account the number of protons of the two species) can be explained by considering additional acetate synthesis from CO₂ by 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.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Synthesis of labeled substrates. The following abbreviations 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."

View larger version (14K): [in this window] [in a new window]   Fig. 3.   1H (400 MHz) and 13C (100 MHz) NMR spectra of sodium acetate (methyl group resonances only) coming from fermentation of 2-phenoxy-[1-2H2]ethanol (3) (A, B) and 2-phenoxy-[2-2H2]ethanol (7) (C, D). For values of coupling constants and isotope shifts, see text.

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 the ¹H 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 (²H(D₂) = 27.0 ppb, C(D₂) = 0.467 ppm) (29, 30), thus indicating the occurrence of CHD₂ and ¹³CHD₂ groups (Fig. 3, C and D). The complete absence of CHD₂-CO and CH₂D-CO species in the product from the former and the latter experiment, respectively, clearly resulted from a comparison 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 feature of this biotransformation appeared to be the shift of a deuterium (hydrogen) atom from carbon-1 to carbon-2 (Reaction 1).

To gain further insight into the process schematized in Reaction 1, samples of 2-phenoxyethanol enriched with ¹³C 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 in ¹H and ¹³C 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 of ¹H and ¹³C nuclei as well as spin-spin coupling constants.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   13C,2H-labeled substrates used in feeding experiments. Compounds 8, 9, and 10 were prepared according to Scheme A of Fig. 2 starting from commercially available Br13CH2COOCH2CH3, BrCH213COOCH2CH3, and Br13CH213COOCH2CH3, respectively.

After fermentation of sample 8, the ¹H NMR spectrum of the resulting acetate showed signals of CH₂D (triplet) and CH₃ at a ratio from which ~45% dilution of the biotransformation product with de novo synthesized acetate could be calculated (neglecting satellite peaks due to ¹³CH₂D and ¹³CH₃ groups). A 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 assumption, the peak intensities measured in the ¹³C NMR spectrum appeared in the expected proportions, i.e. ~1:16:3 for the ¹³CH₃ group (singlet at  26.27, acetate coming from the acetogenic activity of the microorganism), for the ¹³CH₂D group (triplet upfield shifted, acetate coming from the 2-phenoxyethanol supplied), and for the ¹³CO (singlet at  184.39, corresponding to the ¹³C natural abundance level of the whole acetate recovered from the fermentation experiment).

Only the peak due to the [¹³C]carboxylate group was detectable in the ¹³C NMR spectrum of the acetate arising from the bioconversion of 9. The ¹H 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-CO and CH₂D-¹³CO, in the ratio of ~1:2. The absence of the isotopomer CH₂D-CO 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 of Acetobacterium (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, 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, compound 10 was administered to a cell suspension of Acetobacterium after dilution (18 to 100) with unlabeled 2-phenoxyethanol. When the acetate isolated at the end of this fermentation was examined by ¹H NMR (Fig. 5A), no signals assignable to the CH₂D group were observed, 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 containing ¹³CH₃ and ¹³CH₂D groups. The multiplicity 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 CH₃ singlet in agreement with the expected deuterium isotope shift), was indicative of a strong prevalence of ¹³CH₂D-¹³CO species among the [¹³C]methyl isotopomeric mixture (J_HC = 126.7 Hz) (31). The presence of a very minor concentration of ¹³CH₃CO isotopomer was recognizable 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.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   1H (400 MHz) (A) and 13C (50 MHz) NMR spectra (B and C) 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-13CO; b, CH3-13CO; c, 13CH3CO; d, 13CH2D-CO; e, 13CH3-13CO.

These findings were corroborated by the following considerations: (i) by inspection of methyl and carboxyl regions of the ¹³C NMR spectrum (Fig. 5, B and C) the composition of the ¹³C isotopomeric mixture was estimated to be: ¹³CH₂D-¹³CO (a) = 86% (dt at  26.05 and d at  184.40; J_CC = 52 Hz, J_CD = 19.5 Hz) (28, 31); CH₃-¹³CO (b) = 6% (s at  184.42); ¹³CH₃CO (c) = 6% (s at  26.29); ¹³CH₂D-CO (d) 1% (t at  26.05, J_CD); ¹³CH₃-¹³CO (e) 1% (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-CO molecules (measured as the area of the singlet at _H 1.90) and ¹³CH₂D-¹³CO₂ molecules (measured as the total area of the satellite signals, decreased by 1.1% of the CH₃ area 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 isotopomers b and c in the ¹³C isotopomeric mixture was found to be very close to the expected one (±5%) for ¹³C-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 isotopomers d and e, 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the light of a previous report (12) and in light of the results obtained by feeding experiments performed using ¹H- and ¹³C-labeled substrates and resting cell suspensions of Acetobacterium strain 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 features 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 aldehyde 15. 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^·) and then back to C-2 of the product-related radical (13, R = H), generated in turn by a hydroxyl 1,2-shift.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Hypothetical reaction mechanisms for anaerobic glycol ether cleavage. A, commonly accepted reaction mechanism of diol dehydratases (R = H); X· denoting 5'-deoxyadenosyl radical or a protein-based radical. B, putative mechanism of the enzyme-catalyzed C-O cleavage of phenoxyethanol by Acetobacterium sp.; X· denotes a protein-based radical. C, alternative pathway for the conversion of -oxo radical 18 into acetaldehyde; NuH, nucleophile (e.g. H2O).

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 correlation 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 (-carbonyl  enoxy) radical 18 (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 appropriate conformation of the radical anion 17 (40). It is well known that -hydroxy radicals are up to 10⁵ times more acidic than the corresponding alcohols (CH₂OH-^·CHOH has pK_a values of ~10-12) (41). In addition, a base-promoted hydrogen abstraction as schematized in formula 16 is coherent with the marked lowering of gas-phase C-H bond dissociation energy observed when going from 1-alkanols (e.g. 94 ± 2 kcal mol¹ for H-CH₂OH) (42) to alcoholate ions (e.g. 85 kcal mol¹ for H-CH₂O) (43). -Oxo radicals have been proposed as intermediates 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). 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 (XH in Fig. 6B). It can be noted that such a facet of the phenoxyethanol acetaldehyde lyase recalls the reaction mechanism of adenosylcobalamin-dependent ribonucleotide reductase of Lactobacillus 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 mol¹ (43) and bond dissociation energy of H-CH₂COCH₃ was estimated at ~91 kcal mol¹ (45), a temporary addition of a nucleophile to the carbonyl group of the radical 18 might occur (Fig. 6C). This further step would remove the resonance stabilization in 18 (~8 kcal mol¹) (see Table IV, footnote k in Ref. 45), thus facilitating the formation of the C-H bond by the intermediate 19 to give the labile diol 20 (bond dissociation energy for H-CH₂R ~98 kcal mol¹) (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.

	FOOTNOTES

^* 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 defrayed in part by the payment of page charges. The 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, Università 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.

Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M111059200

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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