Stereochemistry of the conversion of 2-phenoxyethanol into phenol and acetaldehyde by Acetobacterium sp.

Article abstract of DOI:10.1002/hlca.200390212

The conversion of 2-phenoxyethanol to phenol and acetate by the anaerobic bacterium Acetobacterium sp. strain LuPhet1 proceeds through acetaldehyde with concomitant migration of a H-atom from C(1) to C(2) of the glycolic moiety. Separate feeding experiments with (R)- and (S)-2-phenoxy(1- ²H)ethanol, prepared via chemoenzymatic syntheses, indicate that the H-atom involved in the 1,2-shift is the pro-S one of the enantiotopic couple of the alcohol function.

Full text of DOI:10.1002/hlca.200390212

Helvetica Chimica Acta Vol. 86 (2003)
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Stereochemistry of the Conversion of 2-Phenoxyethanol into Phenol and
Acetaldehyde by Acetobacterium sp.
by Giovanna Speranza*a), Britta Muellerb), Maximilian Orlandia), Carlo F. Morellia), Paolo Manittoa),
and Bernhard Schinkb)
a
¡
) Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via Venezian 21,
I-20133 Milano (phone: ‡390250314097; fax: ‡390250314072; e-mail: giovanna.speranza@unimi.it)
b) Fakult‰t f¸r Biologie, Universit‰t Konstanz, Universit‰tsstrasse 10, D-78457 Konstanz
The conversion of 2-phenoxyethanol to phenol and acetate by the anaerobic bacterium Acetobacterium sp.
strain LuPhet1 proceeds through acetaldehyde with concomitant migration of a H-atom from C(1) to C(2) of
the glycolic moiety. Separate feeding experiments with (R)- and (S)-2-phenoxy(1-2H)ethanol, prepared via
chemoenzymatic syntheses, indicate that the H-atom involved in the 1,2-shift is the pro-S one of the enantiotopic
couple of the alcohol function.
Introduction. Ether cleavage is particularly difficult both in vitro [1] and in vivo
[2]. For microbial degradation of polyethylene glycol (PEG) molecules (1) both by
aerobic [3] and anaerobic [4] bacteria, several different biochemical mechanisms have
been proposed. However, the formation of a hemi-acetal structure (i.e., 2 and 3) as the
penultimate step of the ether cleavage appears to be the predominant strategy [2]
(Scheme 1).
Scheme 1. Putative Reaction Mechanisms of Microbial PEG Degradation under Aerobic (a) [3] andAnaerobic
(b) [4f] Conditions
The anaerobic homoacetogenic Acetobacterium strain LuPhet1 was found to
degrade low-molecular-weight PEGs, but also to convert 2-phenoxyethanol (4) via
MeCHO (5) to acetate 6 with release of phenol (Scheme 2) [4f]. Thus, 4 can be
regarded as a useful model compound to study the enzymatic ether cleavage of PEG.
Experiments carried out with 2H- and 13C-labeled 2-phenoxyethanol and resting cell
suspensions of strain LuPhet1 allowed us to clarify the fate of the C- and H-atoms of 4
in the reaction giving rise to MeCHO (isolated as AcONa). As shown in Scheme 3,a,

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Scheme 2. Stoichiometry of the Anaerobic Fermentation of 2-Phenoxyethanol by Acetobacterium sp. Strain
LuPhet1
the alcohol function of 2-phenoxyethanol becomes a carboxy group, whereas the
adjacent CH2 group is transformed into a Me group with concomitant intramolecular
1,2-H shift [5]. These findings could be interpreted in terms of 1,2-PhO shift as a route
alternative to the 1,2-OH shift previously hypothesized (see path b of Scheme 1, where
R2 ˆ Ph) [4f]. However, the radical mechanism depicted in Scheme 3,b [5], which does
not involve a hemi-acetal as a necessary intermediate, seems more likely. It is supported
by the well-recognized propensity of ketyl radicals (radical anions) to eliminate
adjacent leaving groups [6] (step II in Scheme 3,b). In addition, it is consistent with a H-
transfer without exchange with the medium [7], and, in this respect, it is reminiscent of
diol-dehydratase-catalyzed reactions [8]. We report here the determination of a
cryptostereochemical feature of the transformation of 2-phenoxyethanol (4) into
MeCHO, namely, the enantioselectivity exerted by the enzyme in the H-abstraction
from the substrate (e.g., reaction I in Scheme 3,b).
Scheme 3. a) Fate of H- andC-Atoms in the Conversion of 2-Phenoxyethanol to AcOH by Strain LuPhet1 .
b) Hypothetical Enzymatic Mechanism for Glycol Ether Cleavage [5].

Helvetica Chimica Acta Vol. 86 (2003)
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Results and Discussion. To distinguish between the enantiotopic H-atoms of the
primary alcohol group of 4, we prepared both the C(1)-monodeuterated enantiomers
of 2-phenoxyethanol, i.e., 7 and ent-7. A substantial amount of (S)-2-phenoxy(1-
2H)ethanol (7) was synthesized by baker×s yeast mediated hydrogenation of the
corresponding deuterated aldehyde 10, which was prepared from ethyl 2-phenoxya-
cetate (8) via the dideuterated alcohol (9; Scheme 4). The enantiomeric purity (ca.
100%) and the D content (monodeuterated molecules > 98%) of the alcohol resulting
1
from the enzymatic reduction were checked by H-NMR of the Mosher ester [9]
(compared with the ester of the racemic mixture, Fig. 1,a and b) and by MS
measurements. The configuration of 7 was expected to be (S) on the basis of the well-
known empirical rules regarding the stereoselective hydrogenation of the carbonyl
group by Saccharomyces cerevisiae (baker×s yeast) [10]. In any case, it was confirmed
by chemical correlation of 7 with (‡)-(S)-2-(benzyloxy)(1-2H)ethanol (13) [11]
(Scheme 5). Compound 13 was obtained through the baker×s yeast mediated reduction
of the deuterated aldehyde 12 and was shown to be identical in all respects (including
the sign and the value of optical rotation) with the compound of unequivocal
configuration previously synthesized by an independent route [11]. The conversion of
13 to 7 was then achieved by exploiting the activation of the alcohol 14 with the
Mitsunobu reagents [12], followed by reaction with phenol to give 15. The inversion of
Scheme 4
a) LiAID4, Et2O. b) Swern oxidation. c) Bakers×s yeast.
Scheme 5
a) EtOH, Py, CH2Cl2. b) LiAID4, Et2O. c) Swern oxidation. d) Baker×s yeast. e) PhCOCl, Py, CH2Cl2. f) H2,
10% Pd/C, MeOH. g) PPh3, diisopropyl azodicarboxylate (DIAD), PhOH, THF. h) NaOH, EtOH.

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Fig. 1. 1H-NMR Signals (300 MHz, CDCl3) due to the glycolic moiety of Mosher×s (R)-esters of a) (S)-2-
phenoxy(1-2H)ethanol; b) rac-2-phenoxy(1-2H)ethanol, and c) (R)-2-phenoxy(1-2H)ethanol
the configuration at C(1) of (S)-2-phenoxy(1-2H)ethanol (7) was accomplished by
means of the classical Mitsunobu procedure [12], thus providing enantiomerically pure
ent-7 in good yields (cf. the Mosher ester in Fig. 1,c).

Helvetica Chimica Acta Vol. 86 (2003)
2633
Fig. 2. 1H- (400 MHz) and 13C-NMR (100 MHz) spectra of AcONa (in NaOD/D2O; Me-group resonances only)
obtainedby fermentation of ( S)-2-phenoxy(1-2H)ethanol (a and b) and( R)-2-phenoxy(1-2H)ethanol (c and d).
For values of coupling constants and isotope shifts, see text.
When Acetobacterium cells (strain LuPhet1) [5] were fed with (S)-2-phenoxy(1-
2H)ethanol (7), the resulting sodium acetate was found to be a mixture of
monodeuterated and nondeuterated molecules in a ratio1) of ca. 2.5 :1. In fact, the
1H- and 13C-NMR spectra of this acetate exhibited the typical patterns of signals due to
CH2D (1:1:1 triplet, 2J(H,D) ˆ 2.05 Hz) and 13CH2D (1:1 :1 triplet, J(C,D) ˆ
2
19.52Hz). These triplets were upfield to the singlets (d(H) ˆ 1.867 ppm, DH(D) ˆ
13.9 ppb; d(C) ˆ 23.593 ppm, DC(D) ˆ 0.235 ppm) due to the nondeuterated Me
group (Fig. 2,a and b) [5]. By contrast, the 1H- and 13C-NMR spectra of sodium acetate
obtained from fermentation of (R)-2-phenoxy(1-2H)ethanol (ent-7) showed only the
signals of the CH3 and 13CH3, respectively, in the Me-group region (Fig. 2,c and d). The
combined results of these feeding experiments were clearly indicative of the capacity of
the enzyme to discriminate between the two H-atoms at C(1) of 4, with consequent
1
1
)
As calculated from the integrated peak areas in the H-NMR spectra, taking into account the number of
H-atoms in the two species.

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migration of the (pro-S) one. It can be noted that, in the acetate arising from the
fermentation of 7, some nondeuterated molecules are present besides the monodeu-
terated ones1). This fact is not due to a partial exchange of the mobile H-atom with the
medium [5], but can be explained by considering that additional CH3COÀ2 is produced
by CO2 reduction by this homoacetogenic bacterium (cf. Scheme 2) [4f][5].
In conclusion, the ether cleavage in the biodegradation of 2-phenoxyethanol (4)
brings about the specific 1,2-shift of one of the two enantiotopic H-atoms at C(1), as
depicted in Scheme 6.
Scheme 6. Stereospecificity of the Microbial Conversion of 2-Phenoxyethanol to AcOH andPhOH
This research was supported by MIUR (Italy) and by Deutsche Forschungsgemeinschaft (Germany) in its
priority program −Radicals in Enzymatic Catalysis×.
Experimental Part
General. TLC: Silica gel 60 F254-precoated aluminum sheets (Merck); detection either by UV or spraying
with a ceric sulfate/ammonium molybdate soln., followed by heating to ca. 1508; eluent, petroleum ether/AcOEt
5 :2. Flash chromatography (FC): silica gel (40 63 mm; Merck); eluent, petroleum ether/AcOEt 5 :2, unless
stated otherwise. GC: Dani 3850 gas chromatograph; injector, 2208; detector (FID), 2208; home-made 2m Â
2mm i.d. glass column, 5% FFAP on Chromosorb W, 80 100 mesh, isothermal analysis at 2008; tR in min.
Optical rotations: Perkin-Elmer 241 polarimeter; 1-dm cell. NMR Spectra: Bruker AC-300 spectrometer at
300.13 (1H), 46.07 (2H), and 75.47 MHz (13C), and Bruker Avance-400 spectrometer at 400.13 (1H) and
100.61 MHz (13C); d in ppm vs. solvent as internal reference (C(H)DCl3: d(H/D) 7.25, d(C) 77.00) or sodium 3-
(trimethylsilyl)(2,2,3,3-2H4)propanoate (d(Me) ˆ 0 ppm) in the case of D2O/NaOD (pH > 10); J in Hz; 13C
multiplicities from APT spectra. EI-MS (m/z [%]): VG 7070 EQ mass spectrometer; at 70 eV.
Fermentation Experiments and Isolation of Sodium Acetate. Labelled 2-phenoxyethanol samples were
transformed by dense cell suspensions of Acetobacterium sp. strain LuPhet1 [5], and the produced acetate was
extracted and prepared as described in [5].
Data of Sodium Acetate. 1H-NMR (400 MHz, D2O/NaOD): 1.8670 (t, 2J(H,D) ˆ 2.05, CH2D); 1.8809
(s, CH3). 13C-NMR (100 MHz, D2O/NaOD): 23.593 (t, J(C,D) ˆ 19.52, CH2D); 23.828 (s, CH3); 181.975
(COO).
(S)-2-Phenoxy(1-2H)ethanol (7). A stirred soln. of oxalyl chloride (1.7 ml, 19.7 mmol) in dry CH2Cl2
(60 ml) was cooled to À 808 under N2 and treated dropwise with DMSO (2.8 ml, 39.4 mmol), keeping the temp.
below À 658. After 15 min, a soln. of 2-phenoxy(1,1-2H2)ethanol (9; 1.4 g, 10 mmol), prepared from ethyl 2-
phenoxyacetate (8) by a published procedure [5], in dry CH2Cl2 (15 ml) was added over a period of 5 min.
Stirring was continued at À 658 for 15 min, then Et3N (6.9 ml, 49.5 mmol) was added dropwise with stirring.
After 10 min, the cooling bath was removed, and the mixture was stirred for 2h at r.t. H 2O (10 ml) was added,
stirring was continued for 10 min, and the two layers were separated. The aq. phase was extracted with CH2Cl2
(2 Â 10 ml), and the org. layers were combined, washed two times with brine, and dried (Na2SO4). Removal of
the solvent under reduced pressure gave the crude 2-phenoxy(1-2H)acetaldehyde (10; 1.35 g, quant. yield) as an
1
oil, which was used immediately in the next step. TLC: Rf 0.35. GC: tR 2.4. H-NMR (300 MHz, CDCl3): 4.59
(s, PhOCH2); 6.87 7.11 (m, 3 arom. H); 7.30 7.41 (m, 2arom. H). 2H-NMR (CHCl3): 9.89 (br. s, 2HÀC(1)).
Compound 10 (675 mg, 4.9 mmol), dissolved in 7 ml of EtOH, was gradually added to a suspension of baker×s
yeast (500 g) in preboiled distilled water (500 ml), and the mixture was vigorously stirred at 378 for 24 h (GC
and TLC control). The fermentation broth was saturated with NaCl and continuously extracted with Et2O. The
Et2O extract was dried (Na2SO4), evaporated under reduced pressure, and purified by FC to give pure 7
25
(400 mg, 58%). TLC: Rf 0.19. GC: tR 4.6. ‰aŠ ˆ ‡0.394 (c ˆ 9.4, CHCl3). 1H-NMR (400 MHz, CDCl3): 2.26 (br.
D
s, OH); 3.92( tt, J ˆ 4.6, 1.8, HÀC(1)); 4.05 (d, J ˆ 4.6, CH2); 6.89 6.97 (m, 3 arom. H); 7.24 7.30 (m, 2arom.

Helvetica Chimica Acta Vol. 86 (2003)
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H). 2H-NMR (CHCl3): 3.91 (br. s, 2HÀC(1)). 13C NMR (100 MHz, CDCl3): 61.06 (t, 1J(C,D) ˆ 21.9, C(1)); 69.01
‡
(CH2); 114.52, 121.07, 129.48 (arom. CH); 158.57 (arom. C). EI-MS: 139 (40, M ), 122 (3), 107 (10), 94 (100);
(2H1) species > 98%. ee was found to be higher than 99% as shown by the NMR spectrum of its (R)-MTPA
ester (see below and Fig. 1,a).
(R)-2-Phenoxy(1-2H)ethanol (ent-7). PPh3 (1.65 g, 6.3 mmol), 4-nitrobenzoic acid (1.05 g, 6.3 mmol) and 7
(294 mg, 2.1 mmol) were dissolved in dry THF/toluene 1:1 (30 ml) under N2. The soln. was cooled to À 208, and
diisopropyl azodicarboxylate (DIAD; 1.24 ml, 6.3 mmol) was added dropwise with stirring over a 5-min period.
After 10 min, the reaction was complete (TLC and GC analysis). Removal of the solvent under reduced
pressure gave a residue, which was dissolved in hexane/AcOEt 2:1 and cooled to 0 8. Insoluble material was
removed by filtration, the filtrate was evaporated under reduced pressure, and the residue was purified by FC to
give pure 2-phenoxy(1-2H)ethyl 4-nitrobenzoate (505 mg, 83%). TLC: Rf 0.49. GC: tR 5.3. 1H-NMR (300 MHz,
CDCl3): 4.33 (d, J ˆ 4.5, CH2); 4.69 (br. t, J ˆ 4.5, HÀC(1)); 6.92 7.00 ( m, 3 arom. H); 7.24 7.32 (m, 2arom.
H); 8.19 8.41 (m, 4 arom. H). 13C-NMR (75 MHz, CDCl3): 61.78 (t, 1J(C,D) ˆ 22.1, C(1)); 63.45 (CH2); 112.49,
121.28, 121.91, 128.62, 129.48 (arom. CH); 133.06, 148.43, 156.23 (arom. C); 162.46 (COO).
To a soln. of the above 2-phenoxy(1-2H)ethyl 4-nitrobenzoate (500 mg, 1.73 mmol) in THF (10 ml) was
added 2n NaOH (3 ml). After vigorous stirring for 2h at r.t., the mixture was diluted with Et 2O and H2O, the
two phases were separated, and the aq. layer was extracted with Et2O. The combined org. extract was dried
(Na2SO4), concentrated, and the residue was purified by FC (petroleum ether/AcOEt 1:1) to give ent-7
25
(211 mg, 88%). Data as for 7, except for optical rotation: ‰aŠ ˆ À0.372( c ˆ 11.0, CHCl3). Enantiomeric purity
D
1
(ee > 99%) was checked by H-NMR spectrum of its (R)-MTPA ester (see below and Fig. 1,c).
rac-2-Phenoxy(1-2H)ethanol. H2O (1 ml) and Amberlyst-15 (300 mg) were added to a soln. of commercial
2-phenoxyacetaldehyde dimethyl acetal (250 mg, 1.37 mmol) in MeCN (10 ml), and the mixture was kept at r.t.
under stirring. After 8 h, the resin was filtered off, and the solvent was evaporated under reduced pressure to
give crude 2-phenoxyacetaldehyde (180 mg, 96%) [13]. TLC: Rf 0.35. GC: tR 2.4. It was dissolved in EtOH
(15 ml), cooled to 08 and treated portionwise with NaBD4 (35 mg, 0.8 mmol) under stirring. The mixture was
allowed to warm to r.t. and stirred for additional 2h. Usual workup and purification by FC (petroleum ether/
AcOEt 1:1) gave pure rac-2-phenoxy(1-2H)ethanol. Data as for 7.
Preparation of MTPA (ˆ 3,3,3-Trifluoro-2-methoxy-2-phenylpropanoic Acid) Esters. The (R)-MTPA esters
of 7, ent-7, and rac-2-phenoxy(1-2H)ethanol were prepared from commercially available (‡)-(S)-MTPA-Cl
according to a published procedure [9]. Usually, 15 mg of the alcohol was used.
(R)-MTPA Ester of 7: TLC: Rf 0.54. 1H-NMR (400 MHz, CDCl3): 3.59 (s, MeO); 4.25 (d, J ˆ 4.8,
PhOCH2); 4.61 (br. t, J ˆ 4.8, CH2HOCO); 6.88 6.91 (m, 2arom. H); 7.02( t, J ˆ 7.2, 1 arom. H); 7.28 7.42
(m, 5 arom. H); 7.56 7.61 (m, 2arom. H). 13C NMR (100 MHz, CDCl3): 55.90 (MeO); 64.42( t, 1J(C,D) ˆ 23.6,
CH2HOCO); 65.57 (CH2); 115.00, 121.80, 127.74, 128.81, 129.97 (arom. CH); 123.66 (q, 1J(C,F) ˆ 288.9); 132.57,
158.57 (arom. C); 166.96 (COO) (see Fig. 1,a).
(R)-MTPA Ester of ent-7: 1H-NMR (400 MHz, CDCl3): 4.77 (br. t, J ˆ 4.8, CH2HOCO) (see Fig. 1,c).
(R)-MTPA Ester of rac-2-Phenoxy(1-2H)ethanol: 1H-NMR (300 MHz, CDCl3): 4.61 (br. t, J ˆ 4.8), 4.77
(br. t, J ˆ 4.8) (CH2HOCO) (see Fig. 1,b).
Preparation of (‡)-(S)-2-(Benzyloxy)(1-2H)ethanol (13). 2-(Benzyloxy)(1-2H)acetaldehyde (12) [11],
prepared from commercial 2-benzyloxyacetyl chloride (11) according to published procedures [5][11], was
submitted to baker×s yeast reduction under the conditions described above for compound 7, giving rise to an oil,
25
D
which was purified by FC to afford 13 [11] (51% overall yield) in pure form. TLC: Rf 0.15. GC: tR 4.8. ‰aŠ
ˆ
20
D
‡0.301 (c ˆ 80, CHCl3; [11]: ‰aŠ ˆ ‡0.387 (neat)). 1H-NMR: as in [11]. 13C-NMR (75 MHz, CDCl3): 61.51
(t, 1J(C,D) ˆ 21.7, C(1)); 71.40, 73.30 (2 CH2); 127.78, 128.47 (arom. CH); 138.00 (arom. C).
Conversion of 13 into 7. Compound 13 was converted to (S)-2-benzyloxy(1-2H)ethyl benzoate in 90% yield
as reported in [11]. This ester (2.1 g, 8.2 mmol) was hydrogenated over 10% Pd/C (1 g) in MeOH (40 ml) at r.t.
for 3 h. Filtration of the catalyst and removal of the solvent under reduced pressure gave (S)-2-hydroxy(1-
2H)ethyl benzoate (14; 1.3 g, 95%). TLC: Rf 0.36. 1H-NMR (300 MHz, CDCl3): 2.93 (br. s, OH); 3.88 (d, J ˆ 4.7,
CH2); 4.37 (br. t, J ˆ 4.7, HÀC(1)); 7.34 7.52( m, 3 arom. H); 7.98 8.02( m, 2arom. H). 13C NMR (75 MHz,
CDCl3): 61.11 (CH2); 66.31 (t, 1J(C,D) ˆ 22.8, C(1)); 128.37, 129.67, 133.15 (arom. CH); 129.84 (arom. C); 167.00
(COO).
A stirred soln. of PPh3 (1.47 g, 5.6 mmol) and diisopropyl azodicarboxylate (DIAD; 1.1 ml, 5.6 mmol) in
THF (60 ml) at 08 was treated, sequentially, with a soln. of freshly distilled PhOH (790 mg, 8.4 mmol) in THF
(4 ml) and then with a soln. of 14 (600 mg, 3.6 mmol) in THF (4 ml) over a period of 15 min. The mixture was
allowed to warm to r.t. and was stirred for an additional 1 h (TLC control). After addition of H2O (3 ml) and a
few drops of conc. HCl, the solvent was removed under reduced pressure. The residue was dissolved in Et2O

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Helvetica Chimica Acta Vol. 86 (2003)
(40 ml), washed with 2n NaOH and with H2O, dried (Na2SO4), and concentrated to ca. a half volume under
reduced pressure. Insoluble materials were removed by filtration, the filtrate was evaporated under reduced
pressure, and the residue was purified by FC to give pure (S)-2-phenoxy(1-2H)ethyl benzoate (15) (600 mg,
68%). TLC: Rf 0.49. 1H-NMR (300 MHz, CDCl3): 4.31 (d, J ˆ 4.8, CH2); 4.66 (br. t, J ˆ 4.8, CH2H); 6.95 7.01
(m, 3 arom. H); 7.25 7.59 (m, 5 arom. H); 8.05 8.10 (m, 2arom. H). 13C NMR (75 MHz, CDCl3): 63.09
(t, 1J(C,D) ˆ 23.0, CH2H); 65.97 (CH2); 114.78, 121.22, 128.36, 129.56, 129.74, 133.07 (arom. CH); 130.02, 158.63
(arom. C); 166.50 (COO).
NaOH in pellets (2.0 g) was added to a soln. of 15 (400 mg, 1.6 mmol) in EtOH (50 ml), and the mixture
was refluxed for 1 h. After cooling to r.t., the solvent was evaporated, and the residue was dissolved in Et2O/H2O
1:1 (40 ml) with stirring. The two layers were separated, and the aq. phase was extracted with Et2O. The org.
phases were combined, dried (Na2SO4), and evaporated under reduced pressure. The residue was purified by
passing through a short column of silica gel (petroleum ether/AcOEt 1 :1), to give (S)-2-phenoxy(1-2H)ethanol
(210 mg, 92%), which was found to be identical to 7 according to the 1H- and 13C-NMR, MS, and optical rotation
data.
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ReceivedMarch 11, 2003