Chemoenzymatic synthesis of optically active alcohol and β-amino-acid derivative containing the difluoromethylene group

Article abstract of DOI:10.1016/j.molcatb.2010.05.009

The bioreduction of α,α-difluorinated ketones, ethyl 2,2-difluoro-3-oxobutanoate (2a) and 2,2-difluoro-1-phenyl-1,3-butanedione (2b), with cells of recombinant Escherichia coli overproducing SCR (Saccharomyces cerevisiae carbonyl reductase from bakers' yeast) and GDH (glucose dehydrogenase from Bacillus megaterium) gave enantiomerically pure alcohols, ethyl (S)-2,2-difluoro-3-hydroxybutanoate ((S)-1a) and (S)-2,2-difluoro-3-hydroxy-1- phenyl-1-butanone ((S)-1b), respectively, in the presence of NADP⁺ and glucose in buffer. The reductions of 2a and 2b proceeded completely at the substrate concentrations of 0.4 M (67 g/L) and 1.0 M (200 g/L), respectively. The opposite enantiomers (R)-1a and (R)-1b were also produced by enzyme E039 (a mixture of carbonyl reductase and formate dehydrogenase) contained in Chiralscreen OH (Daicel Chemical Industries) in the presence of NADH and sodium formate in buffer. Enantiomerically pure (S)-1a was converted by organic synthetic methods into an α,α-difluorinated derivative of (R)-β-aminobutyric acid (BABA) in three steps.

Full text of DOI:10.1016/j.molcatb.2010.05.009

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 198–202
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Chemoenzymatic synthesis of optically active alcohol and ␤-amino-acid
derivative containing the difluoromethylene group
Tadashi Ema∗, Taro Kadoya, Kumiko Akihara, Takashi Sakai∗
Division of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima, Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The bioreduction of ␣,␣-difluorinated ketones, ethyl 2,2-difluoro-3-oxobutanoate (2a) and 2,2-
difluoro-1-phenyl-1,3-butanedione (2b), with cells of recombinant Escherichia coli overproducing SCR
(Saccharomyces cerevisiae carbonyl reductase from bakers’ yeast) and GDH (glucose dehydrogenase from
Bacillus megaterium) gave enantiomerically pure alcohols, ethyl (S)-2,2-difluoro-3-hydroxybutanoate
((S)-1a) and (S)-2,2-difluoro-3-hydroxy-1-phenyl-1-butanone ((S)-1b), respectively, in the presence of
NADP+ and glucose in buffer. The reductions of 2a and 2b proceeded completely at the substrate con-
centrations of 0.4 M (67 g/L) and 1.0 M (200 g/L), respectively. The opposite enantiomers (R)-1a and
(R)-1b were also produced by enzyme E039 (a mixture of carbonyl reductase and formate dehydro-
genase) contained in Chiralscreen OH (Daicel Chemical Industries) in the presence of NADH and sodium
formate in buffer. Enantiomerically pure (S)-1a was converted by organic synthetic methods into an
␣,␣-difluorinated derivative of (R)-␤-aminobutyric acid (BABA) in three steps.
Received 31 March 2010
Received in revised form 20 May 2010
Accepted 22 May 2010
Available online 27 May 2010
Keywords:
Asymmetric reduction
␤-Aminobutyric acid
Fluorine
Whole-cell biotransformation
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
gem-difluorinated compounds are much less common. A few enan-
tioselective chemical methods [22–24], the lipase-catalyzed kinetic
Fluorine plays an important role in pharmaceutical and agro-
chemical compounds [1,2]. Half of the top 10 drugs sold in 2005
and approximately 20% of pharmaceuticals on the market contain
fluorine [3]. A majority of the known organofluorine compounds
bear the trifluoromethyl or fluorine-substituted aromatic groups,
while compounds containing the difluoromethylene group are in
the minority. The difluoromethylene group is isopolar and isos-
teric to the ethereal oxygen atom or the hydroxymethylene group,
which is useful for the creation of biologically active compounds.
For example, certain ␣,␣-difluorinated ketones are known as the
transition-state analogs (inhibitors) of hydrolytic enzymes such as
renins [1].
Only several methods are available for installing the difluo-
romethylene group: the generation and use of difluorocarbene
[4–6], the geminal difluorination of the (thio)carbonyl group
with fluorinating agents such as (diethylamino)sulfur trifluoride
(DAST) [7,8], the replacement of the two hydrogen atoms of
the methylene group with N–F electrophilic fluorinating agents
such as 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane
bis(tetrafluoroborate)(Selectfluor)[9,10], theuseofCF2-containing
building blocks such as BrCF2CO2R [11–18], and the defluori-
nation of trifluoromethyl ketones or trifluoroacetates [19–21].
Furthermore, the methods for the synthesis of optically active
resolution [4,5,25,26], and the asymmetric reduction of ketones
using chiral Ru or Rh complexes [27] or bakers’ yeast [28] have
been reported.
Biocatalysts are useful tools for asymmetric synthesis [29,30].
Among various enzymes, carbonyl reductases are one of the
most popular biocatalysts for preparing optically active alcohols
with high enantiomeric purities [31–39]. We have focused on a
synthetically useful enzyme called SCR (Saccharomyces cerevisiae
carbonyl reductase from bakers’ yeast), which shows broad sub-
strate scope and high enantioselectivity simultaneously [40–45].
The recombinant Escherichia coli overproducing SCR and GDH
(glucose dehydrogenase from Bacillus megaterium, a cofactor-
regenerating enzyme) has been demonstrated to be a powerful tool
for asymmetric synthesis [42–45]. More than 20 ketones have been
converted into the optically active alcohols [42,43], including an
important chiral synthon for clopidogrel, the top 2 drug in 2005
[44,45]. In this paper, we report the chemoenzymatic synthesis of
optically pure ␣,␣-difluorinated alcohols 1a and 1b. In addition, we
also report the conversion of 1a into an ␣,␣-difluorinated derivative
of ␤-aminobutyric acid (BABA) for the first time.
2. Experimental
2.1. Materials
∗
Recombinant E. coli BL21(DE3) cells harboring pESCR and
pABGD, which encode the SCR (S. cerevisiae carbonyl reductase
Corresponding authors. Tel.: +81 86 251 8091; fax: +81 86 251 8092.
E-mail address: ema@cc.okayama-u.ac.jp (T. Ema).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.05.009

T. Ema et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 198–202
199
7.66 (t, J = 7.5 Hz, 1H), 8.05 (d, J = 7.5 Hz, 2H); 13C NMR (CDCl3,
150 MHz) ı 25.0, 111.3 (t, J = 266.6 Hz), 128.9, 130.1, 131.3, 135.1,
187.5 (t, J = 27.6 Hz), 196.0 (t, J = 27.9 Hz); 19F NMR (CDCl3, 282 MHz)
ı −110.1 (s, 2F); IR (neat) 3072, 2976, 2930, 1755, 1699, 1599, 1582,
1450, 1420, 1364, 1296, 1256, 1225, 1151, 1115, 1082, 1053, 964,
from bakers’ yeast) and GDH (glucose dehydrogenase from B. mega-
terium) genes, respectively, were grown as reported previously
[42–45]. Enzyme E039, which is a mixture of carbonyl reductase
and formate dehydrogenase, and Chiralscreen OH kit consisting of
E001, E007, E031, E039, and E078 were provided by Daicel Chem-
ical Industries. NADP+ and NADH were purchased from Oriental
Yeast and Kohjin, respectively. Silica gel column chromatography
was performed using Fuji Silysia BW-127 ZH (100–270 mesh). Thin
layer chromatography (TLC) was performed on Merck silica gel 60
893, 839, 714 cm−1
.
2.1.5. Synthesis of racemic 2,2-difluoro-3-hydroxy-1-phenyl-
1-butanone (1b)
F254
.
2-Chloro-2,2-difluoroacetophenone (0.30 mL, 2.0 mmol) was
added to a mixture of acetaldehyde (1.1 mL, 20 mmol) and acti-
vated zinc dust (0.196 g, 2.99 mmol) in dry DMF (5 mL) under Ar in a
sealed tube at 0 ◦C. The mixture was stirred at room temperature for
20 h. The reaction was quenched with 10% HCl (1 mL), and the mix-
ture was stirred for 30 min. The product was extracted with EtOAc
(3 mL × 4). The mixture was dried over MgSO4, and concentrated.
Purification by silica gel column chromatography (hexane/EtOAc
(10:1)–(5:1)) gave racemic alcohol 1b as a colorless oil (151 mg,
38%) [25]. The spectral data for 1b is shown below (Section 2.2.2).
2.1.1. Synthesis of racemic ethyl 2,2-difluoro-3-
hydroxybutanoate (1a)
Ethyl bromodifluoroacetate (4.0 mL, 31 mmol) was added to
a mixture of acetaldehyde (18 mL, 0.32 mol) and activated zinc
dust (2.63 g, 40.2 mmol) in dry THF (40 mL) in an autoclave. After
the mixture had been stirred at 70 ◦C for 4 h, the reaction was
quenched with 10 drops of saturated aqueous NH4Cl at room tem-
perature. After 30 min, the mixture was dried over MgSO4, and
concentrated. Purification by silica gel column chromatography
(hexane/EtOAc (10:1)–(5:1)) followed by bulb-to-bulb distillation
(10 mmHg, 100 ◦C) gave alcohol 1a as a colorless oil (2.22 g, 43%)
[18]. The spectral data for 1a is shown below (Section 2.2.1).
2.2. Typical procedure for the asymmetric reduction of ketone
with recombinant E. coli
2.1.2. Synthesis of ethyl 2,2-difluoro-3-oxobutanoate (2a)
To a mixture of glucose (1.08 g, 6.00 mmol), NADP+ (10 mg,
12 ␮mol), and E. coli BL21(DE3) cells harboring pESCR and pABGD
(2.0 g) in 0.1 M phosphate buffer (pH 7.0, 10 mL) was added ketone
2 (3.00 mmol). The mixture was stirred in a water bath at 20 ◦C
for 24 h, during which the pH of the solution was kept constant
by adding 2N NaOH. Solid NaCl (5.5 g) was added, and the prod-
uct was extracted with EtOAc (25 mL × 4), where centrifugation
(3,200 rpm, 10 min) was conducted to promote the phase sepa-
ration. The combined organic layers were dried over MgSO4, and
concentrated. Purification by bulb-to-bulb distillation or silica gel
column chromatography gave alcohol 1.
Racemic alcohol 1a (3.53 g, 21.0 mmol) was added dropwise to
a solution of Dess–Martin periodinane (13.4 g, 31.5 mmol) in dry
CH2Cl2 (117 mL) under Ar over 15 min. After the mixture had been
stirred at room temperature for 4 h, the reaction was quenched
with saturated aqueous NaHCO3 (50 mL) and then saturated aque-
ous Na2S2O3 (35 mL). The mixture was stirred for 30 min. The
organic layer was separated, and the product was extracted with
CH2Cl2 (40 mL × 4). The combined organic layers were dried over
MgSO4, and concentrated. Purification by bulb-to-bulb distillation
(15 mmHg, 80 ◦C) gave ketone 2a as a colorless oil (2.53 g, 73%)
[10]: 1H NMR (CDCl3, 600 MHz) ı 1.36 (t, J = 7.2 Hz, 3H), 2.42 (t,
J = 1.8 Hz, 3H), 4.38 (q, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 150 MHz) ı
13.8, 24.1, 63.7, 108.0 (t, J = 263.7 Hz), 161.2 (t, J = 30.6 Hz), 194.8 (t,
J = 29.1 Hz); 19F NMR (CDCl3, 564 MHz) ı −114.9 (d, J = 1.7 Hz, 2F); IR
(neat) 2990, 2943, 1780, 1751, 1420, 1373, 1315, 1234, 1134, 1053,
2.2.1. Ethyl (S)-2,2-difluoro-3-hydroxybutanoate ((S)-1a)
Purification by bulb-to-bulb distillation (17 mmHg, 80 ◦C) gave
alcohol (S)-1a (278 mg, 55%): >99% ee (S); [␣]24 −1.0 (c 1.00,
D
1013, 947, 856, 839, 737 cm−1
.
CHCl3); 1H NMR (CDCl3, 600 MHz) ı 1.35 (dt, J = 0.9, 6.6 Hz, 3H),
1.37 (t, J = 7.2 Hz, 3H), 2.08 (br s, 1H), 4.23 (hept, J = 7.2 Hz, 1H),
4.37 (q, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 150 MHz) ı 13.9, 15.2 (dd,
J = 2.3, 4.1 Hz), 63.1, 68.0 (dd, J = 25.9, 28.3 Hz), 114.5 (dd, J = 254.8,
256.0 Hz), 163.6 (dd, J = 31.1, 32.7 Hz); 19F NMR (CDCl3, 564 MHz) ı
−125.1 (dd, J = 14.7, 265.2 Hz, 1F), −116.7 (dd, J = 6.8, 265.2 Hz, 1F);
IR (neat) 3466, 2991, 2945, 1759, 1448, 1375, 1314, 1217, 1105,
1049, 1015, 907, 843 cm−1; GC: CP-cyclodextrin-␤-2,3,6-M-19 col-
umn (Varian, ꢀ 0.25 mm × 25 m), Inj. 250 ◦C, Col. 80 ◦C, Det. 200 ◦C,
(R) 28.1 min, (S) 30.0 min.
2.1.3. Synthesis of 1-phenyl-3-(1-pyrrolidinyl)-2-butene-1-one
To a solution of 1-phenyl-1,3-butanedione (3.01 g, 18.5 mmol)
in dry CH3CN (46 mL) was added pyrrolidine (1.9 mL, 23 mmol). The
mixture was stirred at room temperature for 22 h. The mixture was
cooled in an ice bath to form a white solid. The solid was collected
by filtration, and the filtrate was concentrated to form a white
solid. The product was recrystallized from CH3CN/hexane to give
the product as a white solid (3.43 g, 86%) [10]: mp 168.1–168.3 ◦C;
1H NMR (CDCl3, 600 MHz) ı 1.99 (br s, 4H), 2.68 (s, 3H), 3.36 (br s,
2H), 3.53 (br s, 2H), 5.60 (s, 1H), 7.36–7.40 (m, 3H), 7.85–7.86 (m,
2H); 13C NMR (CDCl3, 150 MHz) ı 17.9, 24.9, 25.2, 48.1, 48.5, 92.5,
127.2, 127.9, 130.0, 143.0, 161.7, 187.8; IR (KBr) 3021, 2980, 2891,
2.2.2. (S)-2,2-Difluoro-3-hydroxy-1-phenyl-1-butanone ((S)-1b)
Purification by silica gel column chromatography (hex-
ane/EtOAc (10:1)) gave alcohol (S)-1b (447 mg, 74%): >99% ee (S);
2860, 1526, 1427, 1346, 1219, 1157, 1067, 1026 cm−1
.
[␣]28 +25 (c 1.00, CHCl3); 1H NMR (CDCl3, 600 MHz) ı 1.41 (d,
D
J = 6.6 Hz, 3H), 2.55 (br s, 1H), 4.45 (dquint, J = 6.6, 17.4 Hz, 1H),
2.1.4. Synthesis of 2,2-difluoro-1-phenyl-1,3-butanedione (2b)
To a solution of Selectfluor (10.6 g, 29.9 mmol) in dry CH3CN
(280 mL) was added Et3N (1.9 mL, 14 mmol) at −10 ◦C. A solution
of 1-phenyl-3-(1-pyrrolidinyl)-2-butene-1-one (2.93 g, 13.6 mmol)
in dry CH3CN (98 mL) was added dropwise over 20 min. The mixture
was stirred at −10 ◦C for 4.5 h, and silica gel (34 g) was added. The
mixture was stirred at −10 ◦C for 1 h, filtered, and concentrated.
Purification by silica gel column chromatography (hexane/EtOAc
(7:1)) gave 2b as a reddish brown oil (1.56 g, 58%) [10]: 1H NMR
(CDCl3, 600 MHz) ı 2.42 (t, J = 1.5 Hz, 3H), 7.51 (t, J = 7.5 Hz, 2H),
7.51 (t, J = 8.7 Hz, 2H), 7.64–7.67 (m, 1H), 8.12 (d, J = 8.7 Hz, 2H); 13
C
NMR (CDCl3, 150 MHz) ı 14.8 (dd, J = 2.6, 4.4 Hz), 67.5 (dd, J = 24.4,
28.5 Hz), 116.3 (dd, J = 257.1, 261.8 Hz), 128.7, 130.2 (t, J = 3.4 Hz),
132.1, 134.7, 190.5 (dd, J = 30.2, 31.9 Hz); 19F NMR (CDCl3, 564 MHz)
ı −119.9 (dd, J = 18.1, 297.4 Hz, 1F), −109.2 (d, J = 297.4 Hz, 1F); IR
(neat) 3445, 3071, 2991, 2945, 1693, 1599, 1580, 1450, 1286, 1204,
1184, 1144, 1101, 1011, 926, 856, 716, 687, 662 cm−1; HPLC: Chiral-
pak AD-H (Daicel Chemical Industries), hexane/i-PrOH (9:1), flow
rate 0.2 mL/min, detection 254 nm, (S) 34.4 min, (R) 36.7 min.

200
T. Ema et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 198–202
Table 1
Asymmetric reduction of 2 with carbonyl reductases.a
.
Entry
Carbonyl reductase
Ketone
Conv. (%)b
Alcohol 1
2
(mmol)
(M)
(g/L)
Yield (%)c
ee (%)d
1
2
3
4
5
6
7
SCR
SCR
SCR
SCR
SCR
E039
E039
2a
2a
2b
2b
2b
2a
2b
3
4
3
6
10
1
3
0.3
0.4
0.3
0.6
1.0
0.05
0.15
50
67
60
120
200
8
>99
>99
>99
>99
>99
>99
>99
55
61
74
82
84
3e
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
29
77
a
Conditions for entries 1–5: 2 (quantity and concentration indicated above), wet cells of E. coli BL21(DE3) harboring pESCR and pABGD (2.0 g), glucose (2.0 equiv. with
respect to 2), NADP+ (10 mg, 12 ␮mol), 0.1 M phosphate buffer (pH 7.0, 10 mL), 20 ◦C, 24 h. Conditions for entries 6–7: 2 (3.0 mmol), E039 (a mixture of carbonyl reductase and
formate dehydrogenase) contained in Chiralscreen OH (Daicel Chemical Industries, 100 mg), HCO2Na (272 mg, 4.0 mmol), NADH (14 mg, 18 ␮mol), 0.5 M phosphate buffer
(pH 7.0, 20 mL), 20 ◦C, 24 h.
b
Conversion determined by 19F NMR.
Isolated yield of 1.
c
d
Determined by GC (CP-cyclodextrin-␤-2,3,6-M-19 column) for 1a and by HPLC (Chiralpak AD-H, hexane/i-PrOH (9:1)) for 1b.
e
Yield determined by 19F NMR using hexafluorobenzene as an internal standard.
[␣]20 −33.0 (c 1.04, CHCl3); 1H NMR (CDCl3, 400 MHz) ı 1.38 (t,
2.3. General procedure for the asymmetric reduction of ketone
with E039
D
J = 7.2 Hz, 3H), 1.42 (d, J = 7.2 Hz, 3H), 3.93 (sept, J = 7.2 Hz, 1H), 4.38
(q, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz) ı 11.7 (dd, J = 2.3,
4.4 Hz), 13.8, 57.8 (dd, J = 24.5, 28.3 Hz), 63.3, 114.1 (dd, J = 254.0,
257.0 Hz), 162.6 (dd, J = 30.9, 31.9 Hz); 19F NMR (CDCl3, 376 MHz) ı
−120.0 (dd, J = 15.6, 259.8 Hz, 1F), −111.6 (dd, J = 7.7, 259.8 Hz, 1F);
IR (neat) 2991, 2129, 2102, 1771, 1458, 1377, 1308, 1252, 1227,
To a mixture of HCO2Na (272 mg, 4.00 mmol), NADH (14 mg,
18 ␮mol), and Chiralscreen OH E039 (100 mg) in 0.5 M phosphate
buffer (pH 7.0, 20 mL) was added ketone 2 (3.00 mmol). The mixture
was stirred in a water bath at 20 ◦C for 24 h. Alcohol 1 was extracted
and purified as shown above.
1138, 1111, 1065, 1042, 1003, 860 cm−1
.
2.4. Ethyl (S)-3-(trifluoromethanesulfonyloxy)-2,
2.6. Ethyl (R)-3-tert-butoxycarbonylamino-2,
2-difluorobutanoate ((S)-3)
2-difluorobutanoate ((R)-5)
To a solution of (S)-1a (313 mg, 1.86 mmol) in dry CH2Cl2 (14 mL)
under Ar was added dropwise pyridine (1.2 mL, 14.9 mmol) at 0 ◦C
over 5 min. After the mixture had been stirred at 0 ◦C for 15 min,
the resulting mixture was cooled to −40 ◦C. To the mixture was
added dropwise a solution of trifluoromethanesulfonic anhydride
(0.37 mL, 2.25 mmol) in dry CH2Cl2 (5 mL) over 20 min. The mixture
was stirred at −40 ◦C for 4 h. The reaction was quenched with sat-
urated aqueous NaHCO3 at 0 ◦C. The product was extracted with
CH2Cl2 (10 mL × 4). The combined organic layer was dried over
Na2SO4, and concentrated. Purification by silica gel column chro-
matography (hexane/CH2Cl2 (2:1)) gave (S)-3 as a colorless oil
(365 mg, 65%): 1H NMR (CDCl3, 400 MHz) ı 1.39 (t, J = 7.2 Hz, 3H),
1.63 (dt, J = 0.9, 6.8 Hz, 3H), 4.40 (q, J = 7.2 Hz, 2H), 5.28–5.34 (m,
1H); 13C NMR (CDCl3, 100 MHz) ı 13.7, 13.9, 64.1, 80.8 (dd, J = 26.4,
30.9 Hz), 111.4 (dd, J = 254.7, 257.7 Hz), 118.2 (q, J = 317.3 Hz), 161.2
(t, J = 30.9 Hz); 19F NMR (CDCl3, 376 MHz) ı −119.3 (dd, J = 10.7,
271.0 Hz, 1F), −116.6 (dd, J = 8.3, 271.0 Hz, 1F), −76.1 (s, 3F); IR
(neat) 2993, 1778, 1423, 1315, 1219, 1146, 1072, 1042, 1007, 934,
To a solution of (R)-4 (208 mg, 1.08 mmol) in dry EtOH (10 mL)
under Ar was added Boc2O (550 ␮L, 2.40 mmol) and Pd/C (Aldrich,
10% (w/w), 34 mg). The mixture was stirred under H2 at room tem-
perature for 24 h. The mixture was filtered through Celite, and
concentrated. Purification by silica gel column chromatography
(hexane/EtOAc (10:1)) gave (R)-5 as a colorless oil (198 mg, 69%):
98% ee; [␣]20 +7.71 (c 1.02, CHCl3); 1H NMR (CDCl3, 600 MHz) ı
D
1.26 (d, J = 6.6 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.42 (s, 9H), 4.29–4.41
(m, 3H), 4.63 (d, J = 9.0 Hz, 1H); 13C NMR (CDCl3, 150 MHz) ı
13.6, 13.8, 28.1, 48.5 (dd, J = 24.0, 28.7 Hz), 63.0, 80.2, 114.3 (t,
J = 253.8 Hz), 154.6, 163.3 (dd, J = 30.6, 33.0 Hz); 19F NMR (CDCl3,
564 MHz) ı −122.5 (d, J = 255.0 Hz, 1F), −114.9 (d, J = 255.0 Hz,
1F); IR (neat) 3341, 2982, 2939, 1771, 1717, 1524, 1458, 1369,
1308, 1250, 1169, 1069, 1045, 864 cm−1; HRMS (EI) calcd for
C
11H19NO4F2 267.1282, found 267.1283; GC: CP-cyclodextrin-␤-
2,3,6-M-19 column, Inj. 250 ◦C, Col. 110 ◦C, Det. 200 ◦C, (R) 43.5 min,
(S) 44.6 min.
849, 810, 621 cm−1
.
3. Results and discussion
2.5. Ethyl (R)-3-azido-2,2-difluorobutanoate ((R)-4)
In the synthesis of ␣,␣-difluorinated ketones 2a and 2b, we
put a high priority on the isolation of a pure compound. After
trial and error, the Reformatsky reaction of BrCF2CO2Et with
CH3CHO followed by the Dess–Martin oxidation was found to be
a practical method for obtaining pure 2a. On the other hand, the
difluorination of 1-phenyl-3-(1-pyrrolidinyl)-2-butene-1-one with
Selectfluor was a good and easy method for the preparation of pure
2b. The best synthetic procedures are described in the Experimental
section.
To a solution of (S)-3 (342 mg, 1.14 mmol) in dry DMF (6 mL)
under Ar in an ice bath was added NaN3 (88.9 mg, 1.37 mmol). The
mixture was stirred at room temperature overnight. The reaction
was quenched with H2O. The product was extracted with CH2Cl2
(10 mL × 4). The combined organic layer was dried over Na2SO4,
and concentrated. Purification by silica gel column chromatogra-
phy (hexane/EtOAc (5:1)) gave (R)-4 as a yellow oil (181 mg, 82%):

T. Ema et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 198–202
201
7). Curiously, (R)-1a was obtained in only 3% yield for an unknown
reason although the enantiomeric purity was as high as >99% ee
(entry 6). Although we have done the same experiment three times,
the yield was very low in all cases. Because 19F NMR indicated that
the conversion estimated by the ratio of 1a to 2a in the extract was
very high (>99%), we suppose that a side reaction might have pro-
ceeded, such as hydrolysis giving a carboxylic acid that is difficult
to extract. In contrast, (R)-1b was isolated successfully in 77% yield
with >99% ee (entry 7).
␤-Amino acids are important from the viewpoint of biological
activity and synthetic utility. Among them, ␤-aminobutyric acid
(BABA) functions as a partial agonist of glycine receptor in animals,
while BABA participates in a defense mechanism against various
pathogens in plants although the mode of action of BABA has been
clarified only partially [47]. We expected that fluorinated deriva-
tives of BABA might be useful for the elucidation of the mode of
action of BABA in animals or plants, in addition to their synthetic
utility toward other biologically active compounds. In this context,
we decided to convert 1a into a derivative of ␣,␣-difluorinated
BABA 3 (Scheme 1). To the best of our knowledge, no derivatives
of ␣,␣-difluorinated BABA have so far been synthesized. Treatment
of (S)-1a with Tf2O gave (S)-3 in 65% yield, which was then treated
with NaN3 to afford (R)-4 in 82% yield. Subsequent hydrogenation
and in situ protection with Boc2O afforded (R)-5 in 69% yield with
98% ee. Because of the volatility of these compounds, the solvent
was carefully evaporated.
Scheme 1. Synthesis of (R)-5, an ␣,␣-difluorinated derivative of (R)-␤-aminobutyric
acid (BABA).
The bioreduction of ketone 2 was done with recombinant E.
coli coexpressing SCR and GDH [42–45], and alcohol 1 was puri-
fied by bulb-to-bulb distillation or column chromatography. The
results are summarized in Table 1. The absolute configurations of
the obtained alcohols 1a and 1b were determined by the Mosher
method with MTPA [46]. Asymmetric reduction of ␤-keto ester 2a
gave (S)-1a in good yield with >99% ee, and the conversion was com-
plete (>99%) at the substrate concentration of 0.3–0.4 M (entries 1
and 2). The conversion, however, dropped to 84% when the sub-
strate concentration was increased up to 0.5 M (not shown). The
reaction conditions shown in entry 2 therefore seem to be optimal
for the reduction of 2a. On the other hand, the reduction of ␤-
diketone 2b (0.3 M) afforded (S)-1b in 74% yield with >99% ee (entry
3). The less hindered carbonyl group underwent the reduction com-
pletely regioselectively. Because the reaction was completed at the
substrate concentration of 0.6 M (entry 4), the substrate concentra-
tion was increased up to 1.0 M (200 g/L) (entry 5), with the result
that (S)-1b was obtained in 84% yield with >99% ee. This high pro-
ductivity is almost the same as that we have reported previously
[44,45]. At the substrate concentration of 1.3 M, however, the con-
version reached only 36% (not shown).
Asymmetric synthesis of each enantiomer is ideal because these
alcohols 1a and 1b are supposed to be used as chiral building
blocks in future. We therefore searched for enzymes capable of
forming (R)-alcohols. For this purpose, we employed Chiralscreen
OH (Daicel Chemical Industries), a screening kit for selecting an
enzyme suitable for the asymmetric reduction of a target ketone.
We expected that both enantiomers with a high enantiomeric
purity could be obtained much more rapidly and easily by using
this type of enzyme kit than by screening microorganisms [37], the
latter of which contain many carbonyl reductases leading to low
enantioselectivity. The asymmetric reductions of 2a and 2b were
performed at room temperature with E001, E007, E031, E039, or
E078 in Chiralscreen OH according to the supplier’s protocol. The
crude product was extracted with EtOAc and then analyzed directly
by chiral HPLC. As a result, E001, E007, E031, and E078 gave (S)-1a
with >99–95% ee and (S)-1b with >99–27% ee, while only E039 gave
(R)-1a with >99% ee and (R)-1b with 97% ee. Based on these prelim-
inary results, the preparative asymmetric reductions of 2a and 2b
were performed at 20 ◦C with enzyme E039 (Table 1, entries 6 and
4. Conclusions
To the best of our knowledge, no optically active alcohols con-
taining the difluoromethylene group are currently commercially
available. Here we studied the practical chemoenzymatic synthesis
of ethyl 2,2-difluoro-3-hydroxybutanoate (1a) and 2,2-difluoro-
3-hydroxy-1-phenyl-1-butanone (1b), which are expected to be
useful chiral building blocks. The reduction of ethyl 2,2-difluoro-
3-oxobutanoate (2a) and 2,2-difluoro-1-phenyl-1,3-butanedione
(2b) with recombinant E. coli or enzyme E039 (a mixture of car-
bonyl reductase and formate dehydrogenase in Chiralscreen OH
(Daicel Chemical Industries)) gave the corresponding optically pure
alcohols with S or R configurations. In the asymmetric synthesis
of 1b with recombinant E. coli cells harboring pESCR and pABGD,
which encode the SCR (S. cerevisiae carbonyl reductase from bakers’
yeast) and GDH (glucose dehydrogenase from B. megaterium) genes,
respectively, highly enantioselective and regioselective reduction
of 2b proceeded even at a substrate concentration of 1.0 M (200 g/L)
in the presence of NADP+ and glucose in buffer. We expect that
1a can be used as a chiral synthon for ␣,␣-difluoro-␤-amino acids
[1,48,49] while 1b can be used as an analog of 1,3-diol units in
natural products after further reduction to ␣,␣-difluoro 1,3-diol
[50,51]. Here we have converted (S)-1a into a derivative of ␣,␣-
difluorinated BABA, (R)-5, for the first time.
Acknowledgments
We thank Dr. H. Amii (Kobe University) and Dr. M. Kuroboshi
(Okayama University) for valuable advice on the synthesis of diflu-
orinated compounds. We also thank Daicel Chemical Industries for
providing us with Chiralscreen OH. This work was supported by a
Grant-in-Aid for Scientific Research from Japan Society for the Pro-
motion of Science (JSPS). We are grateful to the SC-NMR Laboratory
of Okayama University for the measurement of NMR spectra.
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