Combined batch and continuous flow procedure to the chemo-enzymatic synthesis of biaryl moiety of Odanacatib

Article abstract of DOI:10.1016/j.molcatb.2014.03.017

The development of chemo-enzymatic reaction under continuous flow conditions is an emerging area where biotechnology and organic chemistry can joint efforts in order to develop better process. Here in we report our effort on the development of a continuou

Full text of DOI:10.1016/j.molcatb.2014.03.017

Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Combined batch and continuous flow procedure to the
chemo-enzymatic synthesis of biaryl moiety of Odanacatib
Raquel de Oliveira Lopesa, Amanda S. de Mirandaa, Benedikt Reichartb, Toma Glasnovb,
C. Oliver Kappeb, Robert C. Simonc, Wolfgang Kroutilc, Leandro S.M. Mirandaa,
Ivana C.R. Leala, Rodrigo O.M.A. de Souzaa,∗
a Biocatalysis and Organic Synthesis Group, Instituto de Quimica, Universidade Federal do Rio de Janeiro, Rio de Janeiro 22941-909, Brazil
b Christian Doppler Laboratory for Flow Chemistry and Institute of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
c Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of chemo-enzymatic reaction under continuous flow conditions is an emerging area
where biotechnology and organic chemistry can joint efforts in order to develop better process. Here in
we report our effort on the development of a continuous flow approach to the synthesis of Odanacatib
intermediate using a combined batch and continuous flow apparatus arriving on the desired molecule in
excellent yields and selectivity.
Received 14 January 2014
Received in revised form 28 February 2014
Accepted 21 March 2014
Available online 2 April 2014
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Odanacatib
Flow chemistry
Bioreduction
Continuous process
Cascade reaction
1. Introduction
Odanacatib (1) (Scheme 1) is a potent, orally-active selective
cathepsin K inhibitor, which is currently being developed for the
Osteoporosis is
a
pathological condition characterized by
treatment of postmenopausal osteoporosis [7–9]. Considering the
important role of Odanacatib (1) for the osteoporosis treatment,
the aim of this work is the development of a chemo-enzymatic
synthesis for the chiral biaryl unit 2, by combining enzymatic
reduction and continuous flow methodology, or alternatively batch
microwave chemistry. The retrosynthetic analysis of Odanacatib
(1) shows two main building blocks, the chiral biaryl unit 2 and
the fluorinated amino acid 3 (Scheme 1). Both building blocks have
already been subject of research of several groups [8,10–12], and
our approach consists on the development of a chemo enzymatic
cascade reaction starting with the asymmetric bioreduction of
4ꢀ-bromo-2,2,2-trifluoroacetophenone (4) to the desired (R)-1-(4-
bromophenyl)-2,2,2-trifluoroethanol (5), followed by a continuous
flow Suzuki coupling to afford the desired biaryl unit 2.
increased bone turnover, low bone mass and an increased risk of
fracture. The bone loss results from an imbalance between bone
resorption and formation. Osteoporosis therapies fall into two
classes: antiresorptive drugs, which slow down bone resorption
(e.g. cathepsin K inhibitors) and anabolic drugs, which stimulate
bone formation [1].
The cathepsins are a family of cysteine and aspartic proteases
with collagenolytic activity. Cathepsin K, a cysteine protease, can
efficiently degrade type I and II collagen, both of which are major
matrix components of bone and cartilage. Cathepsin K is the most
abundant cysteine protease expressed in osteoclasts and plays a
central role in mediating bone resorption. During the bone resorp-
tion, cathepsin K is secreted by osteoclasts and accumulates in
the acidified resorption lacunae where it degrades matrix proteins
[2–6].
2. Experimental
2.1. Chemicals and materials
The ketone 1-(4-bromophenyl)-2,2,2-trifluoroethanone (4)
was purchased from commercial sources. The racemic alco-
hol 1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) was synthesized
∗
Corresponding author. Tel.: +55 2125627444.
E-mail addresses: souzarod21@yahoo.com.br, souzarod21@gmail.com
(R.O.M.A. de Souza).
http://dx.doi.org/10.1016/j.molcatb.2014.03.017
1381-1177/© 2014 Elsevier B.V. All rights reserved.

102
R. de Oliveira Lopes et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
Scheme 1. Retrosynthetic analysis of Odanacatib (1).
by conventional reduction of the 1-(4-bromophenyl)-2,2,2-
trifluoroethanone (4) (NaBH4, THF, room temperature) [13]. 1H
NMR and 13C NMR spectra were obtained by using a DPX-300
spectrometer (1H NMR: 300.13 MHz, 13C NMR: 75.5 MHz). Melting
points were determined on a StuartTM SMP3 melting point appa-
ratus. Microwave heating experiments were carried out using a
Monowave 300 single-mode microwave reactor from Anton Paar
GmbH (Graz, Austria). The experiments were performed in a 10 mL
Pyrex microwave process vial equipped with a magnetic stirring
bar at a rate of 600 rpm. Reaction times refer to hold times at the
temperatures indicated and not to total irradiation times. Flow
experiments was performed in an ASIA 110 series microreactor
(Syrris Ltd, Royston, UK), employing a two-feed microreactor stain-
less steel coil [14].
and extraction with ethyl acetate (4 × 5 mL). The organic layer was
dried (Na2SO4) and evaporated at reduced pressure to give the
racemic alcohol 5 as a white powder in 91% yield, mp 52.5–53.6 ◦C
(mp 55–56 ◦C) [9].
1H NMR (300 MHz, CDCl3, TMS) ı (ppm): 2.78 (s, 1H, OH), 5.00
(q, 1H, J = 6 Hz, CHOHCF3), 7.37 (d, 2H, J = 9 Hz, H3 and H5), 7.56 (m,
2H, H2 and H6).
13C NMR (75 MHz, CDCl3, TMS) ı (ppm): 72.5 (q, J = 31.5 Hz,
CHOH), 118.6, 122.3, 126.0 e 129.8 (q, J = 280.5 Hz, CF3), 128.0 (C4),
129.3 (C2 e C6), 132.1 (C3 e C5), 133 (d, C1).
GC-FID: after 1 min at 50 ◦C the temperature was increased in
25 ◦C min−1 steps up to 300 ◦C and kept at 300 ◦C for 4 min. Reten-
tion time: tR (ketone 4) = 5.4 min, tR (alcohol 5) = 6.5 min.
2.5. Asymmetric enzymatic reduction by LB-ADH [15]
2.2. GC–MS analysis
Lyophilized L. brevis cells containing the overexpressed ADH
(LB-ADH) (10 mg) were rehydrated in sodium phosphate buffer
(950 ␮L, 50 mM, pH 7.5) and NADPH (0.74 mg, 1 mM) for
30 min at 30 ◦C while shaking. Then, 1-(4-bromophenyl)-2,2,2-
trifluoroethanone (4) (12.6 mg, 50 mM) and 2-propanol (50 ␮L)
were added to the mixture. Reactions were shaken at 30 ◦C and
700 rpm for 24 h. Then, the reaction was stopped by extraction
with ethyl acetate (4 × 3 mL). The organic layer was separated by
centrifugation (10 min, 5000 rpm) and dried (Na2SO4). Conversions
and enantiomeric excesses of the corresponding alcohols were
determined by GC.
GC–MS analysis was performed on a Trace-GC Ultra – DSQ
II-MS system (ThermoElectron, Waltham, MA, USA). The GC con-
ditions were as follows: HP-5 MS column (30 m × 0.25 mm ID,
0.25 ␮m film, Agilent, Waldbronn, Germany), carrier gas helium
5.0, flow 1 mL/min, temperature gradient identical to GC-FID. The
MS conditions were as follows: positive EI ionization, ionization
energy 70 eV, ionization source temperature 280 ◦C, emission cur-
rent 100 ␮A, full-scan-mode.
2.3. GC-FID analysis
Chiral GC, Chirasil DEX CB column, 140 ◦C to 160 ◦C (1 ◦C min−1),
tR [(R)-1-(4-bromophenyl)-2,2,2-trifluoroethanol] (5) = 10.63 min,
tR [(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanol] (5) = 11.25 min.
GC-FID analysis was performed on
a Trace-GC (Ther-
moFisher) with a flame ionization detector using a HP5 column
(30 m × 0.250 mm × 0.25 ␮m). The detector gas for the flame
ionization is H2 and compressed air (5.0 quality).
2.6. Asymmetric enzymatic reduction by ADH-A [15]
2.4. Synthesis of rac-1-(4-bromophenyl)-2,2,2-trifluoroethanol
(5) [13]
Lyophilized E. coli cells containing the overexpressed ADH-
A
(10 mg or 20 mg) were rehydrated in sodium phosphate
A solution of 1-(4-bromophenyl)-2,2,2-trifluoroethanone (4)
(0.5 g, 2 mmol, 1 equiv.) in 2 mL of tetrahydrofuran was prepared
and cooled in an ice bath. Then, the sodium borohydride (0.037 g,
1 mmol, 0.5 equiv.) was slowly added and the reaction mixture was
stirred at room temperature about 1 h, when the end of reaction was
observed by TLC (Rf = 0.66, eluent: petroleum ether: ethyl acetate
20%, UV in 254 nm). After the solvent volume reduction under
reduced pressure, the isolation was done through the adding water
buffer (850 ␮L, 50 mM, pH 7.5) and NADH (0.71 mg, 1 mM) for
30 min at 30 ◦C while shaking. Then, 1-(4-bromophenyl)-2,2,2-
trifluoroethanone (4) (12.6 mg, 50 mM) and 2-propanol (150 ␮L)
were added to the mixture. Reactions were shaken at 30 ◦C and
700 rpm for different times. Then, the reaction was stopped by
extraction with ethyl acetate (4 × 3 mL). The organic layer was sep-
arated by centrifugation (10 min, 5.000 rpm) and dried (Na2SO4).
Conversions and enantiomeric excesses were determined by GC.

R. de Oliveira Lopes et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
103
The product (R)-1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) was
conversions were determined by GC-FID. The isolated yield was
obtained in 98% yield in 2 h by using 20 mg of ADH-A.
87% (racemic alcohol 2) and 84% (R enantiomer 2).
20
[˛]D −27.5 (c 1.06, EtOH) [9].
Chiral GC, DEX-CB column, 140 ◦C to 160 ◦C (1 ◦C min−1), tR [(R)-
1-(4-bromophenyl)-2,2,2-trifluoroethanol] (5) = 10.63 min, tR [(S)-
1-(4-bromophenyl)-2,2,2-trifluoroethanol] (5) = 11.25 min.
2.10. Asymmetric enzymatic reduction and Suzuki–Miyaura
coupling loading of 5 mol% of palladium under microwave
conditions toward an one-pot process [17]
Lyophilized E. coli cells containing the overexpressed ADH
(ADH-Aꢀ) (40 mg) were added to sodium phosphate buffer
(1700 ␮L, 50 mM, pH 7.5) and NADH (1.42 mg, 1 mM). Then, 1-(4-
bromophenyl)-2,2,2-trifluoroethanone (4) (25.2 mg, 50 mM) was
dissolved in 2-propanol (300 ␮L) and the solution was added to
the mixture. The reaction was shaken at 30 ◦C and 700 rpm for 2 h.
After this time, the supernatant was separated by centrifugation
(10 min, 5000 rpm) and then 4-(methanesulfonyl)phenylboronic
acid (6) (20 mg, 0.1 mmol, 1 equiv.), potassium carbonate (27.6 mg,
0.2 mmol, 2 equiv.) and tetrakis(triphenylphosphine)palladium(0)
(0.23 mg, 0.0002 mmol, 5 mol%) dissolved in isopropanol were
added to the 10 mL Pyrex microwave vial. The reaction was
heated by microwave treatment at 110 ◦C for 5 min. After cool-
ing, the solvent was evaporated and the crude reaction mixture
was extracted with ethyl acetate (4 × 10 mL). The organic layer was
dried (Na2SO4) and conversions were determined by GC-FID. The
conversion in GC-FID was higher than 99% for the product 2.
2.7. Suzuki–Miyaura coupling loading of 0.2 mol% of palladium
under microwave conditions [16]
Reaction optimization on small scale was performed in a
Monowave 300 (Anton Paar GmbH) in 10 mL Pyrex microwave
process vials using standard procedures in temperature control
mode. Suzuki reactions under microwave conditions were carried
out adding 1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) (95.1 mg,
0.375 mmol, 1 equiv.), 4-(methanesulfonyl)phenylboronic acid (6)
(75 mg, 0.375 mmol, 1 equiv.), potassium carbonate (103.6 mg,
0.75 mmol, 2 equiv.), tetrakis(triphenylphosphine)palladium(0)
(0.84 mg, 0.00075 mmol, 0.2 mol%) and a solution of distilled water
and isopropanol (3 mL, 1:1). All the reactions were performed in
closed vessels. After that, the solvent was evaporated and the mix-
ture was extracted with ethyl acetate (4 × 5 mL). The organic layer
was dried (Na2SO4). Conversions were determined by GC-FID and
enantiomeric excesses were determined by HPLC. The product 2
was obtained as a white powder in 92% yield in 5 min at 110 ◦C, mp
171.9–172.8 ◦C.
2.11. Suzuki–Miyaura coupling in continuous flow with catalyst
loading of 5 mol% [16]
1H NMR (300 MHz, DMSO-d6, TMS) ı (ppm): 3.27 (s, 3H,
SO2CH3), 5.25–5.29 (m, 1H, CHOHCF3), 6.95 (d, J = 4.2 Hz, 1H,
CHOH), 7.65 (d, J = 8.4 Hz, 2H, H3 e H5), 7,80 (d, J = 8.4 Hz, 2H, H2
e H6), 7.95–8.04 (m, 4H, H2ꢀ, H6ꢀ, H3ꢀ e H5ꢀ).
An aqueous solution (10 mL) containing 4-(methanesulfonyl)
phenylboronic acid (6) (500 mg, 2.5 mmol, 1 equiv.) and potassium
carbonate (690 mg, 5.0 mmol) and a solution of isopropanol (5 mL)
containing 1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) (317 mg,
1.25 mmol) and tetrakis(triphenylphosphine)palladium(0) (70 mg,
0.2 mol%) were pumped from separate feeds through a coil reactor
(16 mL PFA-coil, 5 min residence time, flow rate 3.2 mL/min) using
a static BPR (7 bar, 100 PSI) in a BPR holder made of steel and heated
at 110 ◦C. The aqueous solution was used in excess to flush the sys-
tem before and after the reaction. The complete reaction mixture
was collected for 10 min and the solvent was evaporated to give
a black residue which was suspended in acetone and filtered. The
filtrate was evaporated and water (20 mL) was added. The mixture
was extracted with ethyl acetate (3 × 20 mL) and the organic layer
was dried (Na2SO4) and evaporated to give a light yellow product
in 73% crude yield. Further purification by column chromatography
furnished the product in 45% yield. Conversions were determined
by GC-FID.
13C NMR (75 MHz, DMSO-d6, TMS) ı (ppm): 43.6 (SO2CH3), 70.1
(q, J = 30.7 Hz, CHOHCF3), 123.2 e 126.9 (d, J = 280.5 Hz, CHOHCF3),
127.1 (C2 and C6), 127.7 (C2ꢀ, C6ꢀ, C3 and C5), 128.4 (C3ꢀ and C5ꢀ),
136.3 (C1ꢀ), 138.9 (C1), 139.8 (C4), 144.6 (C4ꢀ).
2.8. Determination of ee by means of chiral HPLC measurement
Column: Chiralpak AD (Chiracel), length = 25 cm, internal
diameter = 4.6 mm, isocratic flow with 1.0 mL/min, eluent:
n-heptane/2-PrOH = 85:15. tR R-enantiomer (2) = 22.80 min,
tR S-enantiomer (2) = 26.89 min. Determination of conver-
sion by means of achiral GC-FID measurement: after 1 min
at 50 ◦C the temperature was increased in 25 ◦C min−1 steps
up to 300 ◦C and kept at 300 ◦C for 4 min. Retention time: tR
[1-(4-bromophenyl)-2,2,2-trifluoroethanol] (5) = 6.5 min, tR [2,2,2-
trifluoro-1-(4ꢀ-(methylsulfonyl)-[1,1ꢀ-biphenyl]-4-yl)ethanol]
(2) = 11.6 min.
2.12. Synthesis of acetophenones 7, 8 and 10 [16]
2.9. Suzuki–Miyaura coupling in continuous flow loading of
0.2 mol% of palladium [16]
Reactions were performed in a Monowave 300 (Anton
Paar GmbH) in 10 mL Pyrex microwave process vials using
standard procedures in temperature control mode. Suzuki
reactions under microwave conditions were carried out
adding 1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) (95.1 mg,
0.375 mmol, 1 equiv.), corresponding phenylboronic acid
(0.375 mmol, 1 equiv.), potassium carbonate (103.6 mg, 0.75 mmol,
2 equiv.), tetrakis(triphenylphosphine)palladium(0) (0.84 mg,
0.00075 mmol, 0.2 mol%) and a solution of distilled water and
isopropanol (3 mL, 1:1). All the reactions were performed in closed
vessels for 5 min at 110 ◦C. After that, the solvent was evaporated
and the mixture was extracted with ethyl acetate (4 × 5 mL). The
organic layer was dried (Na2SO4). Conversions were determined
by GC-FID.
A
sample of 4-(methanesulfonyl)phenylboronic acid (6)
(250 mg, 1.25 mmol, 1 equiv.) and potassium carbonate (345 mg,
2.5 mmol, 2 equiv.) was dissolved in 5 mL of distilled water.
In an additional flask,
a sample of rac-1-(4-bromophenyl)-
2,2,2-trifluoroethanol (5) (317 mg, 1.25 mmol, 1 equiv.) and
tetrakis(triphenylphosphine)palladium(0) (2.8 mg, 0.0025 mmol,
0.2 mol%) was dissolved in 5 mL of isopropanol. Each reaction
mixture was pumped through the coil reactor (4 mL PFA-coil,
5 min residence time, flow rate 0.8 mL/min) using a static BPR
(7 bar, 100 PSI) in a BPR holder made of steel and heated at 110 ◦C.
The complete reaction mixture was collected for 10 min and the
solvent was evaporated. The mixture was extracted with ethyl
acetate (4 × 10 mL). The organic layer was dried (Na2SO4) and
The product 4ꢀ-(4-methylsulfonylphenyl)-2,2,2-trifluoroaceto-
phenone (7) was obtained as a white powder in 92% yield, mp

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R. de Oliveira Lopes et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
165.7–166.8 ◦C. The melting point is in agreement with previous
report (mp 157.9–158.8 ◦C) [18].
1H NMR (300 MHz, DMSO-d6, TMS) ı (ppm): 3.27 (s, 3H,
SO2CH3), 7.73–7.83 (m, 4H, hydrogen atoms of the aromatic ring),
7.96–8.04 (m, 4H, hydrogen atoms of the aromatic ring).
13C NMR (75 MHz, DMSO-d6, TMS) ı (ppm): 43.6 (SO2CH3), 92.5
(q, JC F = 30.7 Hz, C1), 123.5 (d, JC F = 287.2 Hz, CF3), 126.6 (C2 e C6),
127.7 (C3ꢀ e C5ꢀ), 128.2 (C3 e C5);139.0 (C2ꢀ e C6ꢀ), 139.1 (C1ꢀ), 139.9
(C4ꢀ), 144.5 (C4).
The
product
4ꢀ-(4-methylthiophenyl)-2,2,2-
trifluoroacetophenone (8) was obtained as a yellow powder
in 98% yield, mp 107.2–108.4 ◦C. The melting point is in agreement
with previous report (mp 114.6–116.2 ◦C [18].
Scheme 2. Asymmetric reduction of 4ꢀ-bromo-2,2,2-trifluoroacetophenone (4) to
(R)-1-(4-bromophenyl)-2,2,2-trifluoroethanol (5) by using a single alcohol dehy-
drogenase (ADH-A) and isopropanol as reducing agent to regenerate the cofactor.
1H NMR (300 MHz, CDCl3, TMS) ı (ppm): 2.55 (s, 3H, SCH3), 7.36
(d, 2H, J = 8.55, H3ꢀ e H5ꢀ), 7.59 (d, 2H, J = 8.58, H2ꢀ e H6ꢀ), 7.75 (d,
2H, J = 8.73, H2 e H6), 8.15 (d, 2H, J = 8.64, H3 e H5).
13C NMR (75 MHz, CDCl3, TMS) ı (ppm): 15.7 (s, SCH3), 117 (d,
JC F = 289 Hz, COCF3), 126.9 (C3ꢀ e C5ꢀ), 127.4 (C2ꢀ e C6ꢀ), 127.9 (C3
e C5), 128.6 (C4), 131.1 (d, JC F = 1.5 Hz, C2 e C6), 135.7 (C1ꢀ), 140.5
(C4ꢀ), 147.7 (C1), 180.3 (d, JC F = 35.5 Hz, COCF3).
The product 4ꢀ-phenil-2,2,2-trifluoroacetophenone (10) was
obtained as a white powder in 62% yield, mp 49.8–50.9 ◦C. The
melting point is in agreement with previous report (mp 49–50 ◦C)
[19].
1H NMR (300 MHz, CDCl3, TMS) ı (ppm): 7.46–7.55 (m, 3H, H3ꢀ,
H5ꢀ e H4ꢀ), 7.64–7.68 (m, 2H, H2ꢀ e H6ꢀ), 7.76–7.80 (m, 2H, H2 e H6),
8.16–8.19 (m, 2H, H3 e H5).
13C NMR (75 MHz, CDCl3, TMS) ı (ppm): 117.0 (d, J = 289.5 Hz,
COCF3), 127.6 (C4ꢀ), 127.9 (C2ꢀ e C6ꢀ), 128.8 (C4), 129.2 (C2 e C2ꢀ),
129.4 (C3ꢀ e C5ꢀ), 131.0 (C3 e C5), 139.4 (C1ꢀ), 148.5 (C1), 180.1 (q,
J = 35.2 Hz, COCF3).
2.13. Asymmetric enzymatic reduction of acetophenone 7, 8 and
10 by ADH-A [15]
The asymmetric enzymatic reduction of acetophenones 7, 8 and
10 to corresponding alcohols 2, 9 and 11 was performed as previous
described in Section 2.6.
For the compound (R)-2,2,2-trifluoro-1-(4ꢀ-(methylsulfonyl)-
[1,1ꢀ-biphenyl]-4-yl)ethanol (2), see Section 2.7.
Scheme 3. Asymmetric reduction of 50 mM of acetophenones 7, 8 and 10 to the
alcohol 2, 9 and 11, respectively, by using ADH-A and isopropanol as reducing agent
to regenerate the cofactor. Conversions were determined by GC-FID.
For
the
compound
(R)-2,2,2-trifluoro-1-(4ꢀ-
(methylthio)biphenyl-4-yl)ethanol (9):
1H NMR (300 MHz, CDCl3, TMS) ı (ppm): 2.54 (s, 3H, SCH3), 5.08
(q, 1H, CHOHCF3), 7.33–7.36 (m, 2H, H3ꢀ e H5ꢀ), 7.52–7.56 (m, 4H,
H2, H6, H3 e H5), 7.64–7.61 (m, 2H, H2ꢀ e H6ꢀ).
ketone to the desired alcohol, but also the oxidation of the co-
substrate isopropanol to acetone, thus regenerating the required
cofactor NAD(P)H, which then can be used in catalytic amount
(Scheme 2) [15].
13C NMR (75 MHz, CDCl3, TMS) ı (ppm): 16 (s, SCH3), 72.8 (q,
JC F = 32.2 Hz, CHOHCF3), 124.5 (d, JC F = 285 Hz, CHOHCF3), 127.1
(C3ꢀ e C5ꢀ), 127.2 (C2ꢀ e C6ꢀ), 127.7 (C2 e C6), 128.2 (C3 e C5), 133.0
(C4), 137.3 (C1ꢀ), 138.5 (C4ꢀ), 142.0 (C1).
First, the ADH-A preparation (lyophilized Escherichia coli cells
containing the overexpressed ADH) were rehydrated in sodium
phosphate buffer in the presence of the cofactor NADH. Then, 4ꢀ-
bromo-2,2,2-trifluoroacetophenone (4) dissolved in isopropanol
was added and shaken at 30 ◦C at 700 rpm from 30 min to 6 h. After-
wards, the reaction was stopped by extraction with ethyl acetate
and the organic layer was separated by centrifugation [15]. The GC
analysis showed conversions ≥99% and an enantiomeric excesses
of 98% for the R enantiomer could be obtained after 2 h of reaction
(Table 1). The same reaction profile was also evaluated using 10 mg
of lyophilized cells and similar results were obtained for longer
reaction times (5 h). Isolated yields as high as 98% for the reduc-
tion were achieved. However, increasing the concentration of the
ketone 4 to 100 mM, a slight decrease in conversion was observed
(91% after 24 h).
For the compound (R)-1-(biphenyl-4-yl)-2,2,2-trifluoroethanol
(11)
1H NMR (300 MHz, CDCl3, TMS) ı (ppm): 5.07 (q, 1H, CHOHCF3),
7.37–7.66 (m, 9H, H2, H6, H3, H5, H2ꢀ, H6ꢀ, H3ꢀ, H5ꢀ e H4ꢀ).
13C NMR (75 MHz, CDCl3, TMS) ı (ppm): 72.8 (q, J = 31.5 Hz,
CHOHCF3), 124.5 (d, J = 285 Hz, CHOHCF3), 127.4 (C4ꢀ), 127.6 (C3
e C5), 127.9 (C2ꢀ e C6ꢀ), 128.1 (C2 e C6), 129.1 (C3ꢀ e C5ꢀ), 133.3 (C4),
140.6 (C1), 142.6 (C1ꢀ).
3. Results and discussion
We started our work studying the enzymatic reduction of 4
employing a recombinant alcohol dehydrogenase from Rhodococ-
cus ruber (ADH-A). The enzymatic reduction was carried out
under substrate-coupled cofactor regeneration with isopropanol
as reducing agent. ADH-A catalyzes not only the reduction of the
Other related substrates were also evaluated in the enzy-
matic reduction, including: 2,2,2-trifluoro-1-(4ꢀ-(methylsulfonyl)
biphenyl-4-yl)ethanone (7) and 2,2,2-trifluoro-1-(4ꢀ-(methylthio)
biphenyl-4-yl)ethanone (8). The results indicate low reactivity for

R. de Oliveira Lopes et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
105
Table 1
Asymmetric reduction of a 50 mM solution of 4ꢀ-bromo-2,2,2-trifluoroacetophenone (4) by using ADH-A preparation (lyophilized cells of E. coli containing overexpressed
enzyme – 20 mg).
Entry
Reaction time (h)
Conv. (%) GC-FID
e.e. (%) GC-FID
1
2
3
4
5
0.5
1
2
4
6
74
94
>99
>99
>99
98 (R)
98 (R)
98 (R)
99 (R)
99 (R)
Reactions were shaken at 30 ◦C and 700 rpm. Conversions were determined by GC-FID and enantiomeric excesses were determined by GC-FID.
Table 3
these substrates (Scheme 3), in contrast to 1-(biphenyl-4-yl)-2,2,2-
Suzuki–Miyaura coupling under microwave irradiation at 110 ◦C for 5 min by adding
whole cells, cofactor (NADH), phosphate buffer or phosphate buffer and NADH.
trifluoroethanone (10), which however provided a 96% conversion
by GC-FID, using the same conditions.
Entry
Biotransformation reagent
Conv. (%) GC-FID
Once the bioreduction was successfully achieved, the
next step was the Suzuki–Miyaura coupling between (R)-
1-(4-bromophenyl)-2,2,2-trifluoroethanol (5, 1 equiv.) and
4-(methanesulfonyl)phenylboronic acid (6, 1 equiv.) under con-
trolled microwave heating using potassium carbonate (2 equiv.)
as base, tetrakis(triphenylphosphine)palladium(0) (0.2 mol%) as
catalyst and a solution of distilled water/isopropanol (1:1) as sol-
vent (Scheme 4) [16] in order to use the same solvent system used
in the bioreduction step, aiming to perform a cascade reaction.
Conversions ≥99% toward the desired product were achieved in
5 min at 110 ◦C and provided a 92% isolated yield (Table 2, entries
1–6). Higher conversions were obtained using microwave heating
compared to reactions at room temperature (Table 2, entries 6–7).
The Suzuki–Miyaura coupling was also efficient using different
proportions of water and isopropanol and in the absence of
isopropanol (Table 2, entries 8–10). All reactions occurred without
racemization, therefore product 2 was obtained in the same optical
purity as achieved in the previous bioreductive step.
The optimized reactions under microwave heating were trans-
lated to a continuous flow environment (Asia - Syrris) [20].
Reactants were pumped in separate lines to a T mixer and then
through a PFA-Coil reactor at 110 ◦C (4 mL PFA-Coil, 5 min resi-
dence time, combined flow rate 0.8 mL/min). The reaction mixture
was collected for 10 min, the solvent was evaporated and extracted
with ethyl acetate to give the product in a crude yield of 84%. No
racemization was observed (Scheme 4).
1
2
3
4
E. coli cells only
NADH
Phosphate buffer
Phosphate buffer and NADH
0%
>99%
>99%
>99%
Conversions were determined by GC-FID.
In an effort to improve the overall process efficiency by
decreasing the required number of work-up and purification
steps we attempted to develop a one-pot process consisted
on enzymatic reduction followed by Suzuki–Miyaura coupling
[17]. The asymmetric enzymatic reduction was performed as
described previously and after the end of the first step, 4-
(methanesulfonyl)phenylboronic acid (6, 1 equiv.), potassium
carbonate (2 equiv.), tetrakis(triphenylphosphine)palladium(0)
(0.2 mol%) and isopropanol were added to the reaction mixture in
the 10 mL Pyrex microwave vial. The reaction was then heated by
microwave irradiation at 110 ◦C for 5 min. After cooling, the solvent
was evaporated and the crude reaction mixture was extracted
with ethyl acetate, leading to a GC-FID conversion of only 2% for
the desired Suzuki–Miyaura coupling product (Scheme 5).
In order to find out which factor was disturbing the
Suzuki–Miyaura coupling, the reaction was carried out in the pres-
ence of each one of the biotransformation reagents separately
(Table 3). From those experiments we could conclude that the
whole cells used in the biocatalytic step, but not phosphate buffer or
NADH, were in some way preventing the Suzuki–Miyaura coupling
reaction (Table 3).
Table 2
In an attempt to solve this problem, we carried out a cen-
trifugation after the biocatalytic step, followed by addition of
Suzuki–Miyaura reactants to the supernatant (Scheme 6). Unfor-
tunately, this approach needs high catalyst loadings (≥3 mol%),
to achieve high conversions under microwave heating (Table 4,
entries 1–2). When the Suzuki–Miyaura coupling step was per-
formed at room temperature, however, lower conversions and
longer reaction times were observed even with 5 mol% of palladium
catalyst (Table 4, entry 3 and 4).
The final step was to translate this procedure to a continu-
ous flow environment. Biocatalytic reduction was performed as
described previously and followed by centrifugation and addition
of the Pd catalyst to the supernatant. The supernatant and an aque-
ous solution containing the boronic acid and potassium carbonate
were pumped in separate lines to a T-mixer and then through
Suzuki–Miyaura coupling between (R)-1-(4-bromophenyl)-2,2,2-trifluoroethanol
(5) and 4-(methanesulfonyl)phenylboronic acid (6) under microwave heating and
at room temperature.
Entry
Temperature
Solvent
Reaction time
Conv. (%)
GC-FID
1
2
3
4
5
110 ◦C (MW)
110 ◦C (MW)
80 ◦C (MW)
80 ◦C (MW)
80 ◦C (MW)
r.t.
5 min
1 min
10 min
5 min
1 min
3 h
>99
93
98
95
85
iPrOH: H2O (1:1)
6
60
7
r.t.
7 h
61
8
9
10
H2O
H2O:iPrOH (3:2)
H2O:iPrOH (4:1)
5 min
98
>99
>99
110 ◦C (MW)
Conversions were determined by GC-FID.

106
R. de Oliveira Lopes et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 101–107
Scheme 4. Suzuki–Miyaura coupling in continuous flow. Concentration of alcohol in feed A: 0.25 M. Concentration of boronic acid in feed B: 0.25 M.
were pumped from separate feeds through a PFA-coil reactor at
110 ◦C (4 mL PFA-coil, 5 min residence time, combined flow rate
0.8 mL/min). The reaction mixture was collected for 10 min and full
conversion was observed by GC–MS. The product was obtained in
73% crude yield. Further purification by column chromatography
furnished the product in 45% yield.
Scheme 5. Asymmetric enzymatic reduction using ADH-A and Suzuki–Miyaura
coupling toward an one-pot process.
4. Conclusions
In conclusion we presented an enantioselective two step
synthesis of an important intermediate for the new cathepsin
K inhibitor Odanacatib (1), based on asymmetric biocatalytic
reduction followed by Suzuki–Miyaura cross coupling. It was
demonstrated that a system composed by alcohol dehydrogenase
with substrate-coupled cofactor regeneration leads to the desired
alcohol 5 in excellent yields and enantiomeric excess. In addition,
a Suzuki–Miyaura reaction between 5 and 6 was also achieved
in high yields and translated to a continuous flow process. The
coupling of the biocatalytic reduction with the continuous-flow
Suzuki–Miyaura reaction was developed, leading to the desired
product 2 in 73% overall yield, without isolation of any interme-
diate.
Scheme 6. Asymmetric enzymatic reduction using lyophilized cells from
Escherichia coli (ADH-Aꢀ), centrifugation and Suzuki–Miyaura coupling using the
supernatant.
the PFA-coil reactor at 110 ◦C (16 mL, 5 min residence time, com-
bined flow rate 3.2 mL/min). The reaction mixture was collected for
10 min. Full conversion was observed by GC–MS and the product
was obtained in 73% crude yield and high optical purity.
For comparison purposes, a single Suzuki–Miyaura coupling
reaction with 5 mol% of palladium catalyst was carried out under
continuous flow without performing the previous biocatalytic step.
An aqueous solution containing the boronic acid and base and a
solution of isopropanol containing the halide and metal catalyst
Acknowledgments
Financial support from FAPERJ, CAPES and CNPq – Brazil is
acknowledged. C.O.K acknowledges the Science without Borders
program (CNPq, CAPES) for a “Special Visiting Researcher” fellow-
ship.
Table 4
Asymmetric enzymatic reduction, centrifugation (5000 rpm for 10 min) and
Suzuki–Miyaura coupling under microwave heating (110 ◦C for 5 min) and at room
temperature.
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Pd(PPh3)4
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61
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