Synthesis of enantiopure fluorohydrins using alcohol dehydrogenases at high substrate concentrations

Article abstract of DOI:10.1021/jo400962c

The use of purified and overexpressed alcohol dehydrogenases to synthesize enantiopure fluorinated alcohols is shown. When the bioreductions were performed with ADH-A from Rhodococcus ruber overexpressed in E. coli, no external cofactor was necessary to obtain the enantiopure (R)-derivatives. Employing Lactobacillus brevis ADH, it was possible to achieve the synthesis of enantiopure (S)-fluorohydrins at a 0.5 M substrate concentration. Furthermore, due to the activated character of these substrates, a huge excess of the hydrogen donor was not necessary.

Full text of DOI:10.1021/jo400962c

Note
pubs.acs.org/joc
Synthesis of Enantiopure Fluorohydrins Using Alcohol
Dehydrogenases at High Substrate Concentrations
Wioleta Borzęcka, Ivan Lavandera,* and Vicente Gotor*
́
́ ́ ́
Departamento de Química Organica e Inorganica, University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain
S
* Supporting Information
ABSTRACT: The use of purified and overexpressed alcohol dehydrogenases
to synthesize enantiopure fluorinated alcohols is shown. When the
bioreductions were performed with ADH-A from Rhodococcus ruber
overexpressed in E. coli, no external cofactor was necessary to obtain the
enantiopure (R)-derivatives. Employing Lactobacillus brevis ADH, it was
possible to achieve the synthesis of enantiopure (S)-fluorohydrins at a 0.5 M
substrate concentration. Furthermore, due to the activated character of these
substrates, a huge excess of the hydrogen donor was not necessary.
luorinated compounds have a central role nowadays in
A series of α-fluoroacetophenones (3b−3l) were synthesized
Fpharmaceuticals, agrochemicals, radiotracers, and high- through bromination of the corresponding acetophenones 1
performance materials.1 Particularly, vicinal fluoro alcohols
with N-bromosuccinimide (NBS) and p-toluenesulfonic acid
(p-TsOH) in acetonitrile (MeCN) at 50 °C to obtain the α-
bromo ketones 2,13 followed by nucleophilic substitution using
KF and ZnF2 in MeCN at 100 °C (Scheme 1).14 2-
(fluorohydrins) also present interesting properties as liquid
crystals2 and as precursors of natural product analogues, such as
steroids or carbohydrates,3 since it is well-known that the
introduction of C−F bonds can modulate their physico-
chemical properties, including bioavailability, lipophilicity, and
oxidative stability.
Scheme 1. Synthesis of α-Fluoroacetophenone Derivatives
3a−3l
To obtain these derivatives in a selective fashion, recently
several strategies have been described,4 and among them, the
aperture of racemic or meso-epoxides,5 the C−C formation of
(partially) fluorinated building blocks,6 or the reduction of α-
fluoro ketones7 can be highlighted. Although remarkable
improvements have been made in these fields in the past
years, the use of enzymes8 to synthesize chiral alcohols is very
competitive in terms of selectivity and environmentally friendly
conditions. Thus, in the particular case of the bioreduction of
fluorinated ketone precursors, historically whole cells were
employed to obtain enantioenriched fluorohydrins,9 but due to
the presence of several enzymes with opposite stereopreference,
in many examples, it was not possible to obtain the enantiopure
alcohols. Therefore, in the past few years, the use of isolated or
overexpressed alcohol dehydrogenases (ADHs)10 in combina-
tion with very efficient nicotinamide recycling systems has been
successfully shown to synthesize fluorinated alcohols in
excellent yields and ee.11
Fluoroacetophenone (3a) was prepared as previously
described.15 The synthesis of the corresponding racemic
fluorohydrins was performed by simple treatment of the
ketones with NaBH4 in MeOH at 0 °C.
Because of our previous expertise in ADH-catalyzed
reductions,13,16 we selected a series of purified and overex-
pressed biocatalysts (see the Experimental Section), to achieve
the transformations using ketones 3a−3l as substrates, together
with fluorinated α-bromoacetophenones 2b and 2i, and also
2,2-difluoro- (4) and 2,2,2-trifluoroacetophenone (5) to
Most of the biocatalytic examples achieved until now are
related to the reduction of 2-fluoroacetophenone and 2,2,2-
trifluoroacetophenone,9,11 so due to the relevance of chiral
aromatic fluorohydrins,2,12 we became interested in achieving a
systematic study about the application of purified and
overexpressed ADHs with opposite stereopreference to obtain
both enantiopure antipodes of the fluorohydrin derivatives.
Received: May 6, 2013
Published: June 24, 2013
© 2013 American Chemical Society
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compare the activity and selectivity with the monofluorinated
compound 3a. Buffer Tris-HCl 50 mM pH 7.5 was selected as
an appropriate medium, except for brominated compounds 2b
and 2i, where Tris-H2SO4 was employed to avoid undesired
SN2 reactions.13 Furthermore, 1 mM of the corresponding
nicotinamide cofactor was also added and 2-propanol or
glucose with glucose dehydrogenase (GDH) to recycle it. After
enzymatic screening, overexpressed ADH-A from Rhodococcus
ruber in E. coli (E. coli/ADH-A),17 and commercially available
ADH from Lactobacillus brevis (LBADH)18 were chosen as the
best biocatalysts in terms of activity and selectivity. The
employment of these enzymes is highly desirable since they can
work under the “coupled-substrate” approach using 2-propanol
to recycle the cofactor, diminishing the processing costs and
allowing higher substrate concentrations due to better ketone
solubility in the aqueous buffer. Furthermore, due to the
opposite stereopreference they show, it was possible to get
access to both alcohol enantiomers.
external NADH (Table 2). It was interesting to observe that, in
some cases, the enantiopurity of the final alcohols slightly
Table 2. Bioreductions of Fluorinated Ketones Using E. coli/
ADH-A and 2-PrOH (10% v v−1) without the Addition of
a
External NADH (t = 24 h)
b
cd
,
entry
ketone
concentration (mM)
c (%)
ee (%)
1
2
3
4
5
6
3a
3b
3c
3e
4
200
200
200
200
200
100
>99
>99
>99
>99
>99
>99
>99 (R)
97 (R)
98 (R)
>99 (R)
94 (R)
5
>99 (R)
a
b
For experimental details, see the Experimental Section. Measured by
c
d
GC. Measured by chiral GC. Change in CIP priority.
decreased. This could be probably due to the action of other
NADP-dependent ADHs from the host organism that, in the
absence of additional NADH, could compete with ADH-A,
diminishing the overall selectivity. Moreover, it was demonstra-
ted that, even at high concentrations such as 200 mM,
conversions remained quantitative.
Therefore, substrates 2b, 2i, 3a−3l, 4, and 5 (30 mM) were
subjected under these biocatalytic conditions, obtaining
excellent results with both enzymes (Table 1).
Table 1. Bioreductions of Fluorinated Ketones (30 mM)
a
In a next step, we decided to increase the substrate
concentration, keeping constant the quantity of LBADH in
the presence of 1 mM of the nicotinamide cofactor and just
increasing the amount of 2-PrOH, although, due to the fact that
Using E. coli/ADH-A and LBADH
α-halogenated ketones can be quasi-irreversibly reduced,20
a
huge excess of the hydrogen donor was not necessary. A
selection of the results obtained is shown in Figure 1.
E. coli/ADH-A
LBADH
b
cd
,
b
cd
,
entry
ketone
c (%)
ee (%)
c (%)
ee (%)
1
2
2b
2i
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99
>99
>99
>99
>99
99
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
3
3a
3b
3c
3d
3e
3f
3g
3h
3i
4
5
6
7
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
8
9
10
11
12
13
14
15
16
3j
3k
3l
4
5
a
b
For experimental details, see the Experimental Section. Measured by
c
d
GC. Measured by chiral GC. Change in Cahn−Ingold−Prelog
(CIP) priority.
Gratefully, in every case, the substrate was completely
transformed into the enantiopure (R)- or (S)-alcohol when
using ADH-A or LBADH, independently it bore electron-
withdrawing or electron-donating groups at meta- or para-
positions. Also di- or trifluorinated ketones 4 and 5 afforded
perfect conversions and ee with both biocatalysts.
One additional advantage of working with overexpressed
ADH preparations is the possibility to avoid the use of an
external cofactor when performing the bioreductions.19 There-
fore, some of these ketones were tested at higher substrate
concentrations with E. coli/ADH-A without the addition of
Figure 1. Examples of enantiopure fluorinated alcohols obtained at a
0.5 M substrate concentration with LBADH and 2-PrOH (t = 24 h).
For other results, see the Supporting Information.
Several enantiopure fluorinated alcohols were achieved at a
0.5 M concentration with excellent conversions in the presence
of 3 U of the enzyme and 1 mM NADP+. In these examples,
20% v v−1 of the hydrogen donor 2-PrOH was employed,
approximately a molar excess of 5:1 with regard to the ketone
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using a standard polarimeter and are quoted in units of 10−1 deg cm2
g−1. For new compounds, the indirect assignation of their
configuration was based in two convergent criteria: (a) the perfect
stereoselectivity shown by ADH-A and LBADH for the other
members of this fluorinated family and other similar compounds
and (b) the order of the elution peaks in the chiral GC column.
Synthesis of α-Bromoacetophenone Derivatives 2. To a
solution of the acetophenone derivative (15.0 mmol, 1 equiv) in 8 mL
of acetonitrile were added NBS (2.72 g, 15.3 mmol, 1.02 equiv) and p-
toluenesulfonic acid (2.85 g, 15.0 mmol, 1 equiv). The reaction
mixture was stirred for 24 h at 50 °C. After that time, the solvent was
evaporated under reduced pressure. A water solution of saturated
NaHCO3 (30 mL) was then added, and the solution was extracted
with dichloromethane (3 × 30 mL). The organic layers were
combined and dried over Na2SO4. The solvent was evaporated, and
the residue was subjected to column chromatography (silica gel) using
hexanes/CH2Cl2 (from 9:1 to 4:1) as eluent. These compounds
exhibited physical and spectral data in agreement with those
reported.24 2e (4.30 g, 88% yield), 2i (2.12 g, 66% yield), 2j (2.88
g, 82% yield), 2k (2.45 g, 71% yield), and 2l (2.60 g, 71% yield).
Synthesis of α-Fluoroacetophenone Derivatives 3. A mixture
of KF (4.8 mmol, 1.5 equiv) and ZnF2 (4.8 mmol, 1.5 equiv) in 7 mL
of acetonitrile was stirred and heated at 100 °C in a sealed heavy-
walled Pyrex tube for 1 h. A solution of the substrate (3.2 mmol, 1.0
equiv) in acetonitrile (7 mL) was then added into the reaction
mixture. It was then heated for 24 h at 100 °C. After that time, the
solvent was evaporated under reduced pressure. Water (20 mL) was
then added, and the solution was extracted with dichloromethane (3 ×
20 mL). The organic layers were combined and dried over Na2SO4.
The solvent was evaporated, and the residue was subjected to column
chromatography (silica gel) using hexanes/EtOAc (from 95:5 to
90:10) as eluent. These compounds exhibited physical and spectral
data in agreement with those reported.14,25 3b (230 mg, 47% yield), 3c
(255 mg, 47% yield), 3d (625 mg, 90% yield), 3e (326 mg, 39% yield),
3f (274 mg, 56% yield), 3g (380 mg, 71% yield), 3h (210 mg, 36%
yield), 3i (265 mg, 53% yield), 3j (367 mg, 66% yield), 3k (368 mg,
68% yield), and 3l (214 mg, 36% yield).
substrate, confirming that the bioreduction of these derivatives
was thermodynamically favored.
(R)-6b is a precursor of cholesterol absorption inhibitors
(CAIs), such as AZD4121 or ezetimibe (Scheme 2), a class of
Scheme 2. (R)-6b, a Precursor of Cholesterol Absorption
Inhibitors Such as Ezetimibe
compounds that have attracted attention for the treatment of
hypercholesterolemia, related to several diseases, such as
atherosclerosis.21 This alcohol was recently synthesized via
asymmetric reduction with (R)-2-methyloxazaborolidine in the
presence of borane−dimethyl sulfide complex in THF at 0
°C.22 With regard to biocatalyzed synthesis, Zhu and co-
workers obtained the enantiopure (S)-alcohol using an isolated
ADH from Candida magnolia with glucose and GDH to recycle
the nicotinamide cofactor.23
Because of the relevance of this bromohydrin, the E. coli/
ADH-A catalyzed bioreduction of 2b was performed at a 260
mg scale with a 100 mM substrate concentration, obtaining
enantiopure (R)-6b after purification with 70% yield.
Overall, with the systematic study shown here, it was
demonstrated that ADH-catalyzed hydrogen transfer reduction
of α-fluoro ketones is a very convenient method to synthesize
both alcohol antipodes in an enantiopure form at high substrate
concentrations with excellent conversions. Thus, when
performing the bioreduction in the presence of overexpressed
enzymes, it was also possible to avoid the use of external
nicotinamide cofactor. Because of the nature of these activated
ketones, the employment of a huge excess of the hydrogen
donor was not necessary.
2-Fluoro-4′-iodoacetophenone (3e): White solid; mp 92.4−95.3
1
°C; IR (NaCl) 3054, 2987, 1422, 1265, 972, 896, 741, 666 cm−1; H
NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.4
Hz, 2H), 5.49 (d, J = 46.9 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ
193.0 (d, J = 15.8 Hz), 138.3, 133.0, 129.2 (d, J = 2.9 Hz), 102.3, 83.5
(d, J = 182.5 Hz); 19F NMR (282 MHz, CDCl3) δ −229.9; MS (ESI+,
m/z) 287 [(M + Na)+, 100%], 265 [(M + H)+, 30%]; HRMS (ESI+,
m/z) calculated for C8H6OFINa (M + Na)+, 286.9365; found,
286.9340.
EXPERIMENTAL SECTION
■
General. Acetophenones 1e, 1i, 1j, 1k, and 1l; α-bromoacetophe-
nones 2b, 2c, 2d, 2f, 2g, and 2h; difluorinated ketone 4; and
trifluorinated derivative 5 were obtained from commercial sources. All
other reagents and solvents were of the highest quality available.
LBADH from Lactobacillus brevis and GDH were obtained from
commercial sources. The following overexpressed enzymes in E. coli
have been kindly provided by Prof. Wolfgang Kroutil from the
University of Graz (Austria): ADH-A from Rhodococcus ruber (E. coli/
ADH-A), ADH-T from Thermoanaerobium sp. (E. coli/ADH-T),
SyADH from Sphingobium yanoikuyae (E. coli/SyADH), RasADH from
Ralstonia sp. (E. coli/RasADH), and TeSADH from Thermo-
anaerobacter ethanolicus (E. coli/TeSADH).
3′-Chloro-2-fluoroacetophenone (3j): Yellow oil; IR (NaCl) 3057,
2987, 2933, 1711, 1575, 1428, 1266, 1229, 1101, 1087, 998, 739, 704
1
cm−1; H NMR (300 MHz, CDCl3) δ 7.91 (m, 1H), 7.79 (m, 1H),
7.62 (m, 1H), 7.47 (m, 1H), 5.51 (d, J = 46.8 Hz, 2H); 13C NMR (75
MHz, CDCl3) δ 192.4 (d, J = 16.0 Hz), 135.2 (d, J = 11.6 Hz), 134.1,
130.3, 128.1 (d, J = 2.9 Hz), 126.0 (d, J = 2.7 Hz), 83.6 (d, J = 182.7
Hz); 19F NMR (282 MHz, CDCl3) δ −230.1; MS (ESI+, m/z) 195
[(M + Na)+, 100%], 173 [(M + H)+, 70%]; HRMS (ESI+, m/z)
calculated for C8H6OClFNa (M + Na)+, 194.9992; found, 194.9983.
2-Fluoro-3′-methoxyacetophenone (3k): White solid; mp 55.7−
58.8 °C; IR (NaCl) 3054, 2987, 1710, 1600, 1584, 1432, 1265, 1092,
738, 705 cm−1; 1H NMR (300 MHz, CDCl3) δ 7.42 (m, 3H), 7.18 (m,
1H), 5.54 (d, J = 46.9 Hz, 2H), 3.88 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 193.2 (d, J = 15.2 Hz), 160.0, 134.9, 129.9, 120.7, 120.2 (d, J
= 2.3 Hz), 112.1 (d, J = 2.2 Hz), 83.5 (d, J = 181.4 Hz), 55.5; 19F
NMR (282 MHz, CDCl3) δ −230.8; MS (ESI+, m/z) 191 [(M + Na)+,
100%], 169 [(M + H)+, 85%]; HRMS (ESI+, m/z) calculated for
C9H9O2FNa (M + Na)+, 191.0488; found, 191.0479.
Chemical reactions were monitored by analytical TLC, performed
on silica gel 60 F254 plates, and visualized by UV irradiation. Flash
chromatography was performed using silica gel 60 (230−400 mesh).
Melting points were taken on samples in open capillary tubes and are
uncorrected. IR spectra were recorded on an infrared Fourier
transform spectrophotometer on NaCl pellets. 1H-, 13C, and 19F-
NMR and DEPT were obtained using 300 and 400 MHz
spectrometers for routine experiments. The chemical shifts (δ) are
given in parts per million and the coupling constants (J) in hertz (Hz).
ESI+ mode was used to record mass spectra (MS) and ESI-TOF for
HRMS. Gas chromatography (GC) analyses were performed on a
standard GC chromatograph. Calibration curves were obtained with α-
fluoro ketones and the corresponding fluorohydrins to measure
accurately the enzymatic conversions. Optical rotations were measured
General Procedure for the Synthesis of the Racemic
Alcohols 6−9. To a solution of the corresponding ketone (4.0
mmol) in methanol (5 mL) at 0 °C was added sodium borohydride
(1.2 mmol). When the reduction was completed (according to the
TLC), a few drops of 1 M HCl were added. The solvent was
evaporated under reduced pressure. Water (10 mL) was then added,
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and the solution was extracted with dichloromethane (3 × 10 mL).
The organic layers were combined and dried over Na2SO4. The
solvent was evaporated, and the residue was subjected to column
chromatography (silica gel) using mixtures of hexanes/ethyl acetate
(from 9:1 to 4:1) as eluents. These compounds exhibited physical and
spectral data in agreement with those reported.23,26 Isolated yields:
71−95%.
1H); 13C NMR (75 MHz, CDCl3) δ 148.4, 140.4 (d, J = 7.6 Hz),
132.4, 129.6, 123.3, 121.4, 86.5 (d, J = 174.2 Hz), 71.9 (d, J = 20.5
Hz); 19F NMR (282 MHz, CDCl3) δ −222.7; MS (ESI+, m/z) 208
[(M + Na)+, 100%], 186 [(M + H)+, 45%]; HRMS (ESI+, m/z)
calculated for C8H8NO3FNa (M + Na)+, 208.0374; found, 208.0380;
[α]2D0 = +33.5 (c 1.04, CHCl3), ee >99% (S).
Enzymatic Screening with 3a. In a 1.5 mL Eppendorf vial,
LBADH (3 U) or lyophilized cells of the overexpressed ADH (ADH-
A, ADH-T, RasADH, SyADH, or TeSADH) in E. coli (20 mg) were
added in Tris-HCl buffer 50 mM pH 7.5 (570 μL, 1 mM NADP+ for
LBADH, ADH-T, RasADH, SyADH, and TeSADH or 1 mM NADH
for ADH-A; 1 mM MgCl2 for LBADH) and mixed with 3a (30 mM)
and 2-propanol (30 μL, 5% v v−1, for LBADH, ADH-A, ADH-T,
SyADH, and TeSADH) or with glucose (60 mM) and GDH (5 U) for
RasADH. The reactions were shaken at 30 °C and 250 rpm for 24 h
and stopped by extraction with ethyl acetate (2 × 0.5 mL). The
organic layers were separated by centrifugation (2 min, 13 000 rpm)
and dried over Na2SO4. Conversions of the corresponding alcohols
were determined by GC, obtaining the following results: for LBADH
(>99%), E. coli/ADH-A (>99%), E. coli/RasADH (99%), E. coli/
SyADH (4%), E. coli/ADH-T (99%), and E. coli/TeSADH (64%).
General Procedure for an Enzymatic Reduction Using
LBADH from Lactobacillus brevis. In a 1.5 mL Eppendorf vial,
LBADH (3 U) was added in Tris-HCl buffer 50 mM pH 7.5 (570 μL,
1 mM NADP+, 1 mM MgCl2) and mixed with 2-propanol (30 μL, 5%
v v−1) and the corresponding ketone (30 or 50 mM). The reaction was
shaken at 30 °C and 250 rpm for 24 h and stopped by extraction with
ethyl acetate (2 × 0.5 mL). The organic layer was separated by
centrifugation (2 min, 13 000 rpm) and dried over Na2SO4.
Conversions and ee of the corresponding alcohols were determined
by GC (see Table 1 and Table S1, Supporting Information). For α-
brominated ketones, in order to avoid undesired SN2 reactions with
Tris-HCl buffer,13 Tris-H2SO4 buffer 50 mM pH 7.5 (570 μL, 1 mM
NADP+, 1 mM MgBr2) was used.
General Procedure for an Enzymatic Reduction Using E.
coli/ADH-A from Rhodococcus ruber. In a 1.5 mL Eppendorf vial,
E. coli/ADH-A (20 mg) was added in Tris-HCl buffer 50 mM pH 7.5
(570 μL, 1 mM NADH) and mixed with 2-propanol (30 μL, 5% v v−1)
and the corresponding ketone (30 or 50 mM). The reactions were
shaken at 30 °C and 250 rpm for 24 h and stopped by extraction with
ethyl acetate (2 × 0.5 mL). The organic layer was separated by
centrifugation (2 min, 13 000 rpm) and dried over Na2SO4.
Conversions and ee of the corresponding alcohols were determined
by GC (see Table 1 and Table S1, Supporting Information). For α-
brominated ketones, in order to avoid undesired SN2 reactions with
Tris-HCl buffer,13 Tris-H2SO4 buffer 50 mM pH 7.5 (570 μL, 1 mM
NADH) was used.
2-Bromo-1-(3-fluorophenyl)ethanol (6i): White solid; mp 66.8−
69.0 °C; IR (NaCl) 3054, 2987, 1422, 1265, 1158, 896, 740, 705 cm−1;
1H NMR (300 MHz, CDCl3) δ 7.37 (m, 1H), 7.16 (m, 2H), 7.05 (tdd,
J = 8.4, 2.5, 0.9 Hz, 1H), 4.95 (dd, J = 8.8, 3.3 Hz, 1H), 3.65 (dd, J =
10.5, 3.3 Hz, 1H), 3.55 (dd, J = 10.5, 8.8 Hz, 1H), 2.69 (br s, 1H); 13
C
NMR (75 MHz, CDCl3) δ 163.0 (d, J = 245.3 Hz), 142.8 (d, J = 27.4
Hz), 130.2 (d, J = 31.8 Hz), 121.6 (d, J = 11.4 Hz), 115.3 (d, J = 83.7
Hz), 113.0 (d, J = 89.0 Hz), 73.1, 39.9; 19F NMR (282 MHz, CDCl3)
δ −112.2; [α]2D0 = +41.0 (c 1.13, CHCl3), ee >99% (S).27
1-(4-Chlorophenyl)-2-fluoroethanol (7c):26d [α]D20 = +42.9 (c 1.67,
CHCl3), ee >99% (S).
2-Fluoro-1-(4-iodophenyl)ethanol (7e): Light yellow solid; mp
42.3−45.0 °C; IR (NaCl) 3055, 2987, 1422, 1265, 896, 740, 705 cm−1;
1H NMR (300 MHz, CDCl3) δ 7.73 (d, J = 8.4 Hz, 2H), 7.17 (d, J =
8.2 Hz, 2H), 5.00 (m, 1H), 4.60−4.28 (m, 2H), 2.52 (br s, 1H); 13C
NMR (75 MHz, CDCl3) δ 137.8, 137.7, 128.2, 94.0, 86.8 (d, J = 173.9
Hz), 72.4 (d, J = 20.1 Hz); 19F NMR (282 MHz, CDCl3) δ −229.9;
MS (ESI+, m/z) 289 [(M + Na)+, 100%], 249 [(M − OH)+, 25%];
HRMS (ESI+, m/z) calculated for C8H8OFINa (M + Na)+, 288.9484;
found, 288.9496; [α]2D0 = +30.3 (c 1.04, CHCl3), ee >99% (S).
2-Fluoro-1-(4-methylphenyl)ethanol (7f): White solid; mp 33.8−
37.5 °C; IR (NaCl) 3054, 2987, 1422, 1265, 1090, 1004, 896, 743, 705
1
cm−1; H NMR (300 MHz, CDCl3) δ 7.31 (d, J = 8.1 Hz, 2H), 7.20
(d, J = 8.0 Hz, 2H), 5.02 (m, 1H), 4.61−4.41 (m, 2H), 2.50 (dd, J =
2.9, 0.8 Hz, 1H), 2.38 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 138.3,
135.1 (d, J = 8.2 Hz), 129.3, 126.3, 87.2 (d, J = 173.2 Hz), 72.8 (d, J =
19.6 Hz), 21.2; 19F NMR (282 MHz, CDCl3) δ −220.4; MS (ESI+, m/
z) 177 [(M + Na)+, 100%], 137 [(M − OH)+, 90%]; HRMS (ESI+)
calculated for C9H11OFNa (M + Na)+, 177.0675; found, 177.0686;
[α]2D0 = +51.9 (c 1.34, CHCl3), ee >99% (S).
2-Fluoro-1-(3-fluorophenyl)ethanol (7i): Light yellow oil; IR
(NaCl) 3055, 2986, 1594, 1450, 1266, 1137, 1010, 896, 739, 705
1
cm−1; H NMR (300 MHz, CDCl3) δ 7.37 (m, 1H), 7.16 (m, 2H),
7.05 (tdd, J = 8.4, 2.5, 1.0 Hz, 1H), 5.05 (m, 1H), 4.64−4.31 (m, 2H),
2.57 (br s, 1H); 13C NMR (75 MHz, CDCl3) δ 163.0 (d, J = 245.1
Hz), 140.7 (d, J = 7.5 Hz), 130.2 (d, J = 8.1 Hz), 121.9 (d, J = 2.7 Hz),
115.3 (d, J = 21.0 Hz), 113.4 (d, J = 22.2 Hz), 86.9 (d, J = 173.6 Hz),
72.3 (d, J = 20.1 Hz); 19F NMR (282 MHz, CDCl3) δ −112.4,
−221.5; [α]2D0 = +42.3 (c 1.21, CHCl3), ee >99% (S).27
1-(3-Chlorophenyl)-2-fluoroethanol (7j): Yellow oil; IR (NaCl)
3060, 2983, 2952, 1600, 1576, 1479, 1433, 1266, 1198, 1079, 1012,
Enzymatic Reduction Using E. coli/ADH-A in the Absence of
the Nicotinamide Cofactor. In a 1.5 mL Eppendorf vial, E. coli/
ADH-A (20 mg) was added in Tris-HCl buffer 50 mM pH 7.5 (540
μL) and mixed with 2-propanol (60 μL, 10% v v−1) and the
corresponding ketone (100 or 200 mM). The reactions were shaken at
30 °C and 250 rpm for 24 h and stopped by extraction with ethyl
acetate (2 × 0.5 mL). The organic layer was separated by
centrifugation (2 min, 13 000 rpm) and dried over Na2SO4.
Conversions and ee of the corresponding alcohols were determined
by GC.
1
912, 789, 739, 703 cm−1; H NMR (300 MHz, CDCl3) δ 7.43 (m,
1H), 7.30 (m, 3H), 5.06 (m, 1H), 4.63−4.30 (m, 2H), 2.64 (br s, 1H);
13C NMR (75 MHz, CDCl3) δ 140.1 (d, J = 8.0 Hz), 134.6, 129.9,
128.6, 126.5, 124.5, 86.9 (d, J = 173.7 Hz), 72.3 (d, J = 20.0 Hz); 19F
NMR (282 MHz, CDCl3) δ −221.4; [α]2D0 = +42.1 (c 1.65, CHCl3), ee
>99% (S).27
2-Fluoro-1-(3-methoxyphenyl)ethanol (7k): Light yellow oil; IR
(NaCl) 3056, 2950, 2838, 1603, 1587, 1489, 1467, 1456, 1436, 1320,
1
1267, 1158, 1043, 1010, 881, 786, 737, 700 cm−1; H NMR (300
Enzymatic Reduction of Ketones at 100 or 200 mM
Concentration Using LBADH. In a 1.5 mL Eppendorf vial,
LBADH (3 U) was added in Tris-HCl buffer 50 mM pH 7.5 (540
μL, 1 mM NADP+, 1 mM MgCl2) and mixed with 2-propanol (60 μL,
10% v v−1) and the corresponding ketone (100−200 mM). The
reactions were shaken at 30 °C and 250 rpm for 24 h and stopped by
extraction with ethyl acetate (2 × 1 mL). The organic layer was
separated by centrifugation (2 min, 13 000 rpm) and dried over
Na2SO4. Conversions and ee of the corresponding alcohols were
determined by (chiral) GC (see Table S1, Supporting Information).
For α-brominated ketones, in order to avoid undesired SN2 reactions
with Tris-HCl buffer,13 Tris-H2SO4 buffer 50 mM pH 7.5 (540 μL, 1
mM NADP+, 1 mM MgBr2) was used.
MHz, CDCl3) δ 7.31 (m, 1H), 6.97 (m, 2H), 6.89 (m, 1H), 5.02 (m,
1H), 4.63−4.32 (m, 2H), 3.84 (s, 3H), 2.39 (br s, 1H); 13C NMR (75
MHz, CDCl3) δ 159.9, 139.7 (d, J = 8.2 Hz), 129.7, 118.5, 113.9,
111.8, 87.2 (d, J = 173.3 Hz), 72.9 (d, J = 19.7 Hz), 55.3; 19F NMR
(282 MHz, CDCl3) δ −220.7; MS (ESI+, m/z): 193 [(M + Na)+,
100%], 153 [(M − OH)+, 50%]; HRMS (ESI+, m/z) calculated for
C9H11O2FNa (M + Na)+, 193.0637; found, 193.0635; [α]2D0 = +42.3 (c
1.85, CHCl3), ee >99% (S).
2-Fluoro-1-(3-nitrophenyl)ethanol (7l): Light yellow solid; mp
49.9−54.8 °C; IR (NaCl) 3054, 2987, 1534, 1422, 1353, 1265, 896,
738, 705 cm−1; 1H NMR (300 MHz, CDCl3) δ 8.32 (ap t, J = 2.0 Hz,
1H), 8.22 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.59
(ap t, J = 8.0 Hz, 1H), 5.16 (m, 1H), 4.69−4.36 (m, 2H), 2.76 (br s,
7315
dx.doi.org/10.1021/jo400962c | J. Org. Chem. 2013, 78, 7312−7317

The Journal of Organic Chemistry
Note
and 19F-NMR for new compounds are described. This material
is available free of charge via the Internet at http://pubs.acs.org.
Enzymatic Reduction of Ketones at 100 or 200 mM
Concentration Using E. coli/ADH-A. In a 1.5 mL Eppendorf vial,
E. coli/ADH-A (20 mg) was added in Tris-HCl buffer 50 mM pH 7.5
(540 μL, 1 mM NADH) and mixed with 2-propanol (60 μL, 10% v
v−1) and the corresponding ketone (100−200 mM). The reactions
were shaken at 30 °C and 250 rpm for 24 h and stopped by extraction
with ethyl acetate (2 × 1 mL). The organic layer was separated by
centrifugation (2 min, 13 000 rpm) and dried over Na2SO4.
Conversions and ee of the corresponding alcohols were determined
by (chiral) GC (see Table S1, Supporting Information). For α-
brominated ketones, in order to avoid undesired SN2 reactions with
Tris-HCl buffer,13 Tris-H2SO4 buffer 50 mM pH 7.5 (540 μL, 1 mM
NADH) was used.
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 985 103452, +34 985 103448. Fax: +34 985 103448.
E-mail: lavanderaivan@uniovi.es (I.L.), vgs@uniovi.es (V.G.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
W.B. thanks the Principado de Asturias for her predoctoral
fellowship (Severo Ochoa Program). I.L. thanks the Spanish
■
Enzymatic Reduction of Ketones at 0.5 M Concentration
Using LBADH. In a 1.5 mL Eppendorf vial, LBADH (3 U) was added
in Tris-HCl buffer 50 mM pH 7.5 (480 μL, 1 mM NADP+, 1 mM
MgCl2) and mixed with 2-propanol (120 μL, 20% v v−1) and the
corresponding ketone (0.5 M). The reaction was shaken at 30 °C and
250 rpm for 24 h and stopped by extraction with ethyl acetate (2 × 0.5
mL). The organic layer was separated by centrifugation (2 min, 13 000
rpm) and dried over Na2SO4. Conversions and ee of the corresponding
alcohols were determined by GC (see Figure 1 and Table S1,
Supporting Information). For α-brominated ketones, in order to avoid
undesired SN2 reactions with Tris-HCl buffer,13 Tris-H2SO4 buffer 50
mM pH 7.5 (480 μL, 1 mM NADP+, 1 mM MgBr2) was used.
Scale-up of the Bioreductions Using LBADH. In a 10 mL glass
vial, LBADH (15 U) was added in Tris-HCl buffer 50 mM pH 7.5 (1
mM NADP+, 1 mM MgCl2) and mixed with 2-propanol (10% v v−1)
and the corresponding ketone (50 mg, 100 mM). The reaction was
shaken at 30 °C and 250 rpm for 24 h and stopped by extraction with
diethyl ether (2 × 5 mL). The organic layer was dried over Na2SO4,
and the solvent was carefully evaporated (Caution! Fluorohydrins are
highly volatile). Conversions and ee of the corresponding alcohols were
determined by GC and NMR. For 2i, in order to avoid undesired SN2
reactions with Tris-HCl buffer,13 Tris-H2SO4 buffer 50 mM pH 7.5 (1
mM NADP+, 1 mM MgBr2) was used. In most cases, the final
enantiopure products exhibited excellent purity after solvent
evaporation, so no further purification was necessary. To obtain (S)-
6i, (S)-7f, and (S)-7j, flash chromatography was employed to purify
the alcohol derivatives. (S)-6i (37 mg, 73% yield, >99% ee), (S)-7a (47
mg, 93% yield, >99% ee), (S)-7b (44 mg, 87% yield, >99% ee), (S)-7c
(36 mg, 71% yield, >99% ee), (S)-7d (46 mg, 91% yield, >99% ee),
(S)-7e (44 mg, 87% yield, >99% ee), (S)-7f (33 mg, 64% yield, >99%
ee), (S)-7g (48 mg, 94% yield, >99% ee), (S)-7h (48 mg, 95% yield,
>99% ee), (S)-7i (42 mg, 83% yield, >99% ee), (S)-7j (36 mg, 71%
yield, >99% ee), (S)-7k (41 mg, 81% yield, >99% ee), (S)-7l (45 mg,
88% yield, >99% ee), (S)-8 (42 mg, 83% yield, >99% ee), and (S)-9
(43 mg, 85% yield, >99% ee).
Ministerio de Ciencia e Innovacion
́
(MICINN) for personal
funding (Ramon y Cajal Program). Financial support of this
́
work by the Spanish MICINN (Project MICINN-12-
CTQ2011-24237) is gratefully acknowledged.
REFERENCES
■
(1) (a) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chem.
Soc. Rev. 2008, 37, 320−330. (b) Ametamey, S. M.; Honer, M.;
Schubiger, P. A. Chem. Rev. 2008, 108, 1501−1516.
(2) Kusumoto, T.; Hiyama, T. In Enantiocontrolled Synthesis of Fluoro-
Organic Compounds: Stereochemical Challenges and Biomedicinal
Targets; Soloshonok, V. A., Ed.; Wiley: Chichester, 1999; pp 535−556.
(3) For some recent examples, see: (a) Cresswell, A. J.; Davies, S. G.;
Lee, J. A.; Morris, M. J.; Roberts, P. M.; Thomson, J. E. J. Org. Chem.
2012, 77, 7262−7281. (b) Chen, J.-L.; Zheng, F.; Huang, Y.-E.; Qing,
F.-L. J. Org. Chem. 2011, 76, 6525−6533.
(4) (a) Hollingworth, C.; Gouverneur, V. Chem. Commun. 2012, 48,
2929−2942. (b) Furuya, T.; Kamlet, A. S.; Ritter, T. Nature 2011, 473,
470−477. (c) Haufe, G. J. Fluorine Chem. 2004, 125, 875−894.
(5) Kalow, J. A.; Doyle, A. G. J. Am. Chem. Soc. 2011, 133, 16001−
16012.
(6) Arroyo, Y.; Sanz-Tejedor, M. A.; Parra, A.; García Ruano, J. L.
Chem.Eur. J. 2012, 18, 5314−5318.
(7) Mohanta, P. K.; Davis, T. A.; Gooch, J. R.; Flowers, R. A., II J.
Am. Chem. Soc. 2005, 127, 11896−11897.
(8) (a) Drauz, K., Groger, H., May, O., Eds. Enzyme Catalysis in
̈
Organic Synthesis, 3rd ed.; Wiley-VCH: Weinheim, 2012. (b) Faber, K.
Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin, 2011.
(9) Some selected examples: (a) Barkakaty, B.; Takaguchi, Y.;
Tsuboi, S. Tetrahedron 2007, 63, 970−976. (b) Nakamura, K.;
Yamanaka, R. Tetrahedron: Asymmetry 2002, 13, 2529−2533.
(c) Matsuda, T.; Harada, T.; Nakajima, N.; Itoh, T.; Nakamura, K.
J. Org. Chem. 2000, 65, 157−163.
Scale-up of the Bioreduction of Ketone 2b Employing E.
coli/ADH-A. In a 25 mL Erlenmeyer flask, E. coli/ADH-A (460 mg)
was added in Tris-H2SO4 buffer 50 mM pH 7.5 (10.8 mL, 1 mM
NADH) and mixed with 2-propanol (1.2 mL, 10% v v−1) and ketone
2b (260 mg, 100 mM). Reactions were shaken at 30 °C and 250 rpm
for 24 h. After that time, the pellet was separated by centrifugation (15
min, 6000 rpm) and extracted with ethyl acetate (3 × 20 mL). The
organic layers were combined and dried over Na2SO4. The solvent was
evaporated, and the crude was purified by flash chromatography.
Isolated yield of (R)-6b: 184 mg, 70%, >99% ee.
(10) Recent bibliography: (a) Hollmann, F.; Arends, I. W. C. E.;
Holtmann, D. Green Chem. 2011, 13, 2285−2313. (b) García-Urdiales,
E.; Alfonso, I.; Gotor, V. Chem. Rev. 2011, 111, PR110−PR180.
(c) Musa, M. M.; Phillips, R. S. Catal. Sci. Technol. 2011, 1, 1311−
1323.
(11) (a) Xu, G.-C.; Yu, H.-L.; Zhang, X.-Y.; Xu, J.-H. ACS Catal.
2012, 2, 2566−2571. (b) Itoh, N.; Isotani, K.; Nakamura, M.; Inoue,
K.; Isogai, Y.; Makino, Y. Appl. Microbiol. Biotechnol. 2012, 93, 1075−
1085. (c) Wang, L.-J.; Li, C.-X.; Ni, Y.; Zhang, J.; Liu, X.; Xu, J.-H.
Bioresour. Technol. 2011, 102, 7023−7028. (d) Grau, B. T.; Devine, P.
N.; DiMichele, L. N.; Kosjek, B. Org. Lett. 2007, 9, 4951−4954.
(12) Schirlin, D.; Baltzer, S.; VanDorsselaer, V.; Weber, F.; Weill, C.;
Altenburger, J. M.; Neises, B.; Flynn, G.; Rimy, J. M.; Tarnus, C.
Bioorg. Med. Chem. Lett. 1993, 3, 253−258.
(13) Bisogno, F. R.; Cuetos, A.; Orden, A. A.; Kurina-Sanz, M.;
Lavandera, I.; Gotor, V. Adv. Synth. Catal. 2010, 352, 1657−1661.
(14) Adapted from: Zizhan, C.; Zhu, W.; Zheng, Z.; Zou, X. J.
Fluorine Chem. 2010, 131, 340−344.
(15) Mąkosza, M.; Bujok, R. J. Fluorine Chem. 2005, 126, 209−216.
(16) (a) Cuetos, A.; Bisogno, F. R.; Lavandera, I.; Gotor, V. Chem.
Commun. 2013, 49, 2625−2627. (b) Cuetos, A.; Rioz-Martínez, A.;
2-Bromo-1-(4-fluorophenyl)ethanol (6b):23 1H NMR (300 MHz,
CDCl3) δ 7.39 (m, 2H), 7.08 (ap t, J = 8.7 Hz, 2H), 4.93 (m, 1H),
3.65−3.50 (m, 2H), 2.72 (d, J = 3.2 Hz); 13C NMR (75 MHz, CDCl3)
δ 162.6 (d, J = 245.3 Hz), 136.0 (d, J = 3.0 Hz), 127.7 (d, J = 8.3 Hz),
115.6 (d, J = 21.4 Hz), 73.2, 40.1; 19F NMR (282 MHz, CDCl3) δ
−113.4.
ASSOCIATED CONTENT
■
S
* Supporting Information
Enzymatic conversions and ee at higher substrate concen-
trations (Table S1), analytical methods, and copies of 1H-, 13C-,
7316
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The Journal of Organic Chemistry
Note
Bisogno, F. R.; Grischek, B.; Lavandera, I.; de Gonzalo, G.; Kroutil,
W.; Gotor, V. Adv. Synth. Catal. 2012, 354, 1743−1749. (c) Kurina-
Sanz, M.; Bisogno, F. R.; Lavandera, I.; Orden, A. A.; Gotor, V. Adv.
Synth. Catal. 2009, 351, 1842−1848.
(17) This (S)-selective enzyme has a strong preference for NADH/
NAD+. See: Edegger, K.; Gruber, C. C.; Poessl, T. M.; Wallner, S. R.;
Lavandera, I.; Faber, K.; Niehaus, F.; Eck, J.; Oehrlein, R.; Hafner, A.;
Kroutil, W. Chem. Commun. 2006, 2402−2404.
(18) This (R)-selective enzyme shows a preference for NADPH/
NADP+. See, for instance: Wolberg, M.; Hummel, W.; Wandrey, C.;
Muller, M. Angew. Chem., Int. Ed. 2000, 39, 4306−4308.
̈
(19) (a) Ma, H.; Yang, L.; Ni, Y.; Zhang, J.; Li, C.-X.; Zheng, G.-W.;
Yang, H.; Xu, J.-H. Adv. Synth. Catal. 2012, 354, 1765−1772. (b) Shen,
N.-D.; Ni, Y.; Ma, H.-M.; Wang, L.-J.; Li, C.-X.; Zheng, G.-W.; Zhang,
J.; Xu, J.-H. Org. Lett. 2012, 14, 1982−1985.
(20) Bisogno, F. R.; García-Urdiales, E.; Valdes
Kroutil, W.; Suar
ez, D.; Gotor, V. Chem.Eur. J. 2010, 16, 11012−
11019.
́
, H.; Lavandera, I.;
́
(21) Burnett, D. A. Curr. Med. Chem. 2004, 11, 1873−1887.
(22) Karlsson, S.; Sorensen, J. H. Org. Process Res. Dev. 2012, 16,
̈
586−594.
(23) Ren, J.; Dong, W.; Yu, B.; Wu, Q.; Zhu, D. Tetrahedron:
Asymmetry 2012, 23, 497−500.
(24) (a) Macharla, A. K.; Nappunni, R. C.; Marri, M. R.; Peraka, S.;
Nama, N. Tetrahedron Lett. 2012, 53, 191−195. (b) Duca, M.;
Malnuit, V.; Barbault, F.; Benhida, R. Chem. Commun. 2010, 46,
6162−6164. (c) Richards, J. J.; Melander, C. J. Org. Chem. 2008, 73,
5191−5193. (d) Pravst, I.; Zupan, M.; Stavber, S. Tetrahedron 2008,
64, 5191−5199.
(25) Raja, E. K.; Klumpp, D. A. Tetrahedron Lett. 2011, 52, 5170−
5172.
(26) (a) Castro, L. C. M.; Bezier, D.; Sortais, J.-B.; Darcel, C. Adv.
Synth. Catal. 2011, 353, 1279−1284. (b) Beier, P.; Alexandrova, A. V.;
Zibinsky, M.; Surya Prakash, G. K. Tetrahedron 2008, 64, 10977−
10985. (c) Fuglseth, E.; Sundby, E.; Bruheim, P.; Hoff, B. H.
Tetrahedron: Asymmetry 2008, 19, 1941−1946. (d) Liu, J.; Ni, C.; Li,
Y.; Zhang, L.; Wang, G.; Hu, J. Tetrahedron Lett. 2006, 47, 6753−6756.
(e) Stavber, G.; Zupan, M.; Jereb, M.; Stavber, S. Org. Lett. 2004, 6,
4973−4976. (f) Sibille, S.; Mcharek, S.; Perichon, J. Tetrahedron 1989,
45, 1423−1428.
(27) It was not possible to measure the MS spectra of this compound
since it decomposed in all conditions tried.
7317
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