Mechanism of Anaerobic Ether Cleavage
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111059200
ISSN1083-351X
AutoresGiovanna Speranza, Britta Mueller, Maximilian Orlandi, Carlo F. Morelli, Paolo Manitto, Bernhard Schink,
Tópico(s)Chemical Reactions and Isotopes
Resumo2-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 α-hydroxyradical and of its conjugate base (ketyl anion) to eliminate a β-positioned leaving group. 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 α-hydroxyradical and of its conjugate base (ketyl anion) to eliminate a β-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.White G.F. Russel N.J. Tidswell E.C. Microbiol. Rev. 1996; 60: 216-232Crossref PubMed Google Scholar). An important group of xenobiotic ether compounds, the linear polyether PEG 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity 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.Cox D.P. Adv. Appl. Microbiol. 1978; 23: 173-194Crossref PubMed Scopus (56) Google Scholar). PEGs were found to be degraded by various bacteria, both in the presence and the absence of molecular oxygen (aerobically, Refs. 2.Cox D.P. Adv. Appl. Microbiol. 1978; 23: 173-194Crossref PubMed Scopus (56) Google Scholar, 3.Pearce B.A. Heydeman M.T. J. Gen. Microbiol. 1980; 118: 21-27Google Scholar, 4.Thélu J. Medina L. Pelmont J. FEMS Microbiol. Lett. 1980; 8: 187-190Crossref Scopus (15) Google Scholar, 5.Kawai F. Crit. Rev. Biotechnol. 1987; 6: 273-307Crossref Scopus (97) Google Scholar, 6.Obradors N. Aguilar J. Appl. Environ. Microbiol. 1991; 57: 2383-2388Crossref PubMed Google Scholar; anaerobically, Refs. 7.Schink B. Stieb M. Appl. Environ. Microbiol. 1983; 45: 1905-1913Crossref PubMed Google Scholar, 8.Dwyer D. Tiedje J.M. Appl. Environ. Microbiol. 1983; 46: 185-190Crossref PubMed Google Scholar, 9.Grant M.A. Payne W.J. Biotechnol. Bioeng. 1983; 25: 627-630Crossref PubMed Scopus (20) Google Scholar, 10.Wagener S. Schink B. Appl. Environ. Microbiol. 1988; 54: 561-565Crossref PubMed Google Scholar, 11.Schramm E. Schink B. Biodegradation. 1991; 2: 71-79Crossref PubMed Scopus (29) Google Scholar, 12.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar). 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.White G.F. Russel N.J. Tidswell E.C. Microbiol. Rev. 1996; 60: 216-232Crossref PubMed Google Scholar). 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.Frey P.A. Reed G.H. Arch. Biochem. Biophys. 2000; 382: 6-14Crossref PubMed Scopus (33) Google Scholar, 14.Toraya T. Cell. Mol. Life Sci. 2000; 57: 106-127Crossref PubMed Scopus (143) Google Scholar, 15.Smith D.M. Golding B.T. Radom L. J. Am. Chem. Soc. 2001; 123: 1664-1675Crossref PubMed Scopus (90) Google Scholar). 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.Schink B. Stieb M. Appl. Environ. Microbiol. 1983; 45: 1905-1913Crossref PubMed Google Scholar, 10.Wagener S. Schink B. Appl. Environ. Microbiol. 1988; 54: 561-565Crossref PubMed Google Scholar, 11.Schramm E. Schink B. Biodegradation. 1991; 2: 71-79Crossref PubMed Scopus (29) Google Scholar, 16.Strass A. Schink B. Appl. Microbiol. Biotechnol. 1986; 25: 37-42Crossref Scopus (26) Google Scholar, 17.Frings J. Schramm E. Schink B. Appl. Environ. Microbiol. 1992; 58: 2164-2167Crossref PubMed Google Scholar). polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity 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.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar) 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.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar). With this, the EG-degrading enzyme behaved identically to the known diol dehydratases (18.Toraya T. Sigel H. Sigel A. Metal Ions in Biological Systems. 30. Marcel Dekker Inc., New York1994: 217-254Google Scholar). Cleavage of 2-phenoxyethanol, on the other hand, was influenced neither by various corrinoids, including adenosylcobalamin, nor by light (12.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar), 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 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. 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 (δ) are given in parts per million and were referenced to the signals of CDCl3 (δH 7.25 and δC 77.00 ppm) or to 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (δMe 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.Kalinowski H.-O. Berger S. Braun S. Carbon-13 NMR Spectroscopy. John Wiley & Sons, Chichester, UK1988: 47-51Google Scholar). 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. 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.Widdel F. Pfennig N. Arch. Microbiol. 1981; 129: 395-400Crossref PubMed Scopus (594) Google Scholar) with 10 mm2-phenoxyethanol as sole organic carbon substrate under a N2/CO2 atmosphere (80:20 v/v) as described previously (12.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar). 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 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. 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 δH 1.88–2.03 (literature 1.90), δC23.8–26.3 (literature 23.97), and δC 182.0–184.4 (literature 182.02) (21.Gottlieb H.E. Kotlyar V. Nudelman A. J. Org. Chem. 1997; 62: 7512-7515Crossref PubMed Scopus (2862) Google Scholar) for the methyl and the carbonyl group, respectively. This substrate was prepared according to Ref. 22.Ciommer B. Schwarz H. J. Organomet. Chem. 1983; 244: 319-328Crossref Scopus (14) Google Scholar 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 (Rf 0.61) and GC (tR 6.8 min), 1H NMR and EIMS as in Ref. 23.Ogawa, T., Hikasa, T., Ikegami, T., Ono, N., and Suzuki, H. (1994) J. Chem. Soc. Perkin Trans. I 13473–3478Google Scholar; 13C NMR as in Ref. 24.Takeuchi Y. Itoh N. Koizumi T. Yamagami C. Takeuchi Y. Magn. Reson. Chem. 1992; 30: 58-64Crossref Scopus (6) Google Scholar. 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 compound3 (142 mg, 92% yield) pure by GC (tR8.5 min); 1H NMR and EIMS as in Ref. 22.Ciommer B. Schwarz H. J. Organomet. Chem. 1983; 244: 319-328Crossref Scopus (14) Google Scholar; 13C NMR (CDCl3, 50 MHz) δ 60.71 (CD2, quintet, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 21.4 Hz), 68.96 (CH2), 114.54 (CH), 121.07 (CH), 129.45 (CH), 158.57 (C). polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity 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 (Rf 0.46, eluent as above), 1H, and 13C NMR (25.Solladiè G. Colobert F. Denni D. Tetrahedron Asymmetry. 1998; 9: 3081-3094Crossref Scopus (23) Google Scholar). 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 (Rf 0.15, eluent as above) and GC (tR 8.7 min), which was used for the next step without further purification; 1H NMR and EIMS as in Ref. 26.Hammerschmidt, F. (1988) Liebigs Ann. Chem. 537–542Google Scholar; 13C NMR (CDCl3, 50 MHz) δ 61.11 (CD2, quintet, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 21.9 Hz), 71.35 (CH2), 73.30 (CH2), 127.81 (CH), 128.47 (CH), 138.00 (C). polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity 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) δ 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) δ 67.02 (CD2, quintet, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 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) δ 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) δ 61.17 (CH2), 68.34 (CD2, quintet, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 21.9 Hz); EIMS m/z (rel. int.) 140 (M+, 25), 109 (10.Wagener S. Schink B. Appl. Environ. Microbiol. 1988; 54: 561-565Crossref PubMed Google Scholar), 94 (100), 77 (35). polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity These substances were obtained using differently13C-labeled ethyl bromoacetate and LiAlD4according to the procedure described above for 2-phenoxy-[1-2H2]ethanol (3). Ethyl phenoxy-[2-13C]acetate: 1H NMR (CDCl3, 200 MHz) δ 1.29 (t, 3H, J = 7.1 Hz), 4.27 (q, 2H, J = 7.1 Hz), 4.61 (d, 2H, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCH = 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 compound8: 1H NMR (CDCl3, 200 MHz) δ 1.97 (s, 1H, OH), 4.08 (s, 1.84H and d, 0.16H, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCH = 143.3 Hz), 6.91–7.01 (m, 3H), 7.26–7.33 (m, 2H); 13C NMR (CDCl3, 50 MHz) δ 68.98 (13CH2). Ethyl phenoxy-[1-13C]acetate: 1H NMR (CDCl3, 200 MHz) δ 4.27 (dq, 2H,3 JCH = 3.1 Hz, J = 7.1 Hz), 4.61 (d, 2H, 2 JCH = 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) δ 4.08 (s, 2H); 13C NMR (CDCl3, 50 MHz) δ 60.84 (13CD2, quintet, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 21.4 Hz); EIMS m/z (rel. int.) 141 (M+, 20), 107 (10.Wagener S. Schink B. Appl. Environ. Microbiol. 1988; 54: 561-565Crossref PubMed Google Scholar), 94 (100), 77 (35). Ethyl phenoxy-[1,2-13C2]acetate: 1H NMR (CDCl3, 200 MHz) δ 4.27 (dq, 2H,3 JCH = 3.1 Hz, J = 7.1 Hz), 4.61 (dd, 2H, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCH = 146.0 Hz,2 JCH = 4.7 Hz); 13C NMR (CDCl3) δ 65.48 (13CH2, d, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCC = 64.5 Hz), 169.12 (13CO, d, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCC = 64.5 Hz); EIMS m/z (rel. int.) 182 (M+, 90), 108 (100), 94 (25), 77 (80); it gave 10: 1H NMR (CDCl3) δ 4.08 (d, 2H, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCH = 142.8 Hz); 13C NMR (CDCl3) δ 60.98 (13CD2, doublet of quintets, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCC = 39.3 Hz, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCD = 21.4 Hz), 69.08 (13CH2, d, 1The abbreviations used are: PEGpolyethylene glycolEGethylene glycolTLCthin layer chromatographyFCflash chromatographyGCgas chromatographyEIMSelectron impact mass spectrometryrel. int.relative intensity JCC = 39.3 Hz); EIMS m/z (rel. int.) 142 (M+, 20), 108 (5.Kawai F. Crit. Rev. Biotechnol. 1987; 6: 273-307Crossref Scopus (97) Google Scholar), 94 (100), 77 (35). polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity polyethylene glycol ethylene glycol thin layer chromatography flash chromatography gas chromatography electron impact mass spectrometry relative intensity 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.Ciommer B. Schwarz H. J. Organomet. Chem. 1983; 244: 319-328Crossref Scopus (14) Google Scholar) (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 of13C) 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 of1H and 13C NMR signals due to the CH2D and 13CH2D groups. In both cases, this pattern consists of a 1:1:1 triplet (27.Vederas J.C. Nat. Prod. Rep. 1987; 4: 277-337Crossref PubMed Google Scholar) (2 JHD = 2.09 Hz, JCD = 19.5 Hz) (28.Hommeltoft S.I. Baird M.C. J. Am. Chem. Soc. 1965; 107: 2548-2549Crossref Scopus (23) Google Scholar, 29.Matta M.S. Broadway D.E. Stroot M.K. J. Am. Chem. Soc. 1987; 109: 4916-4918Crossref Scopus (9) Google Scholar), which is upfield with respect to the non-deuterated methyl group (2ΔH(D) = 13.5 ppb, ΔC(D) = 0.254 ppm) (29.Matta M.S. Broadway D.E. Stroot M.K. J. Am. Chem. Soc. 1987; 109: 4916-4918Crossref Scopus (9) Google Scholar, 30.Hansen P.E. Annual Reports on NMR Spectroscopy. 15. Academic Press, London1983: 105-234Google Scholar). 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.Frings J. Schink B. Arch. Microbiol. 1994; 162: 199-204Crossref PubMed Scopus (13) Google Scholar) (Fig. 1). In addition, a partial loss of both deuterium atoms during the conversion of 2-phenoxyethanol into phenol and acetate could not be exclude
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