Lipase-catalyzed asymmetric acylation of boron cluster-containing secondary alcohols

Article abstract of DOI:10.1016/j.tetasy.2014.10.009

The lipase-catalyzed asymmetric acetylation of secondary alcohols containing a carborane (boron cluster) moiety was investigated. Most lipases examined showed poor catalytic activity toward carborane-containing secondary alcohol 1a, but lipase TL efficiently catalyzed the acetylation of 1a with high enantioselectivity, to afford (R)-3a. This selectivity is similar to that of the general lipase-catalyzed acylation of secondary alcohols. Utilizing lipase TL, we succeeded in the resolution of carborane-containing alcohol 5, synthesized as a progesterone receptor ligand candidate, and evaluated the activities of the two enantiomers.

Full text of DOI:10.1016/j.tetasy.2014.10.009

Tetrahedron: Asymmetry 25 (2014) 1505–1512
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Lipase-catalyzed asymmetric acylation of boron cluster-containing
secondary alcohols
Shuichi Mori a, Ryohei Takagaki a, Shinya Fujii a, Mio Matsumura b, Aya Tanatani b, Hiroyuki Kagechika a,
⇑
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 August 2014
Accepted 10 October 2014
The lipase-catalyzed asymmetric acetylation of secondary alcohols containing a carborane (boron cluster)
moiety was investigated. Most lipases examined showed poor catalytic activity toward carborane-con-
taining secondary alcohol 1a, but lipase TL efficiently catalyzed the acetylation of 1a with high enantiose-
lectivity, to afford (R)-3a. This selectivity is similar to that of the general lipase-catalyzed acylation of
secondary alcohols. Utilizing lipase TL, we succeeded in the resolution of carborane-containing alcohol
5, synthesized as a progesterone receptor ligand candidate, and evaluated the activities of the two
enantiomers.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
lipases act selectively on the (R)-enantiomer of secondary alcohols
to afford acylated products by discriminating between ‘large’ and
Carboranes (more specifically, dicarba-closo-dodecaboranes,
C2B10H12) are icosahedral boron clusters with a bulky spherical
surface. There are three isomers, which involve the positional rela-
tionship of the two carbon atoms, namely o-, m-, and p-carboranes.
Carboranes have remarkable thermal and chemical stability, and
exceptionally high hydrophobicity,1 and have been employed as
hydrophobic core structures of bioactive molecules in the field of
medicinal chemistry. We have developed several potent carbo-
rane-based ligands of nuclear receptors, such as retinoid recep-
tors,2 estrogen receptor,3 androgen receptor,4 progesterone
receptor,5 and vitamin D receptor.6 Most carborane-containing
bioactive molecules that have been developed so far are achiral
or racemic. In the case of vitamin D derivatives, the optically active
side chain was introduced onto the carborane cage, and the bind-
ing structure of each enantiomer was discussed.6a Therefore, the
development of efficient synthetic methods to prepare optically
active carborane derivatives is of interest in order to develop novel
modulators of various physiological targets and to investigate the
structure–activity relationships in detail.
‘small’ substituents at the stereogenic center.8 However, there
are structural limitations on the substrates of the lipases that can
be effectively converted. For example, enzymatic asymmetric acyl-
ation of 1-(1-adamantyl)ethanol has been reported, but either the
reactivity was very low9 or an unusual enzyme had to be used.10
Therefore, it is of interest to know whether or not bulky carboranes
can be recognized by lipases and, if they can, how they are recog-
nized. Herein we examined the catalytic potency of lipases toward
carborane-containing secondary alcohols, with the aim of develop-
ing an efficient method for the preparation of enantiomerically
pure carborane derivatives.
2. Results and discussion
2.1. Design and synthesis of model substrates
Secondary alcohols with an aromatic group as a ‘large’ substitu-
ent, such as 1-phenylethanol or 1-naphthylethanol, have been
investigated in detail as model substrates for lipase-catalyzed
asymmetric acylation. On the other hand, we have shown that phe-
One method to obtain optically active molecules is to use an
enzyme-catalyzed enantioselective reaction. For example, lipases
have been used for the kinetic resolution of optically active alco-
hols and carboxylic acids because they possess broad substrate
specificity and high enantioselectivity.7 It is well established that
nylcarborane is
a useful core structure of nuclear receptor
ligands.3–5 Here we have designed 1-(7-phenyl-1,7-dicarba-closo-
dodecaboran-1-yl)ethanol 1a as a model substrate for investigat-
ing the lipase-catalyzed asymmetric acylation of carboranes
(Fig. 1). In order to clarify the structural requirements of carborane
derivatives as lipase substrates, we also synthesized compounds
1b–1d.
⇑
Corresponding author. Tel.: +81 3 5280 8032.
E-mail address: kage.chem@tmd.ac.jp (H. Kagechika).
http://dx.doi.org/10.1016/j.tetasy.2014.10.009
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

1506
S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
by using n-butyl lithium as a base and acetaldehyde, propanal, or
OH
OH
OH
propylene oxide as an electrophile to afford racemic 1a (72%), 1b
(69%), and 1d (28%), respectively. Treatment of m-carborane with
1.1 equiv of n-butyllithium, followed by benzaldehyde, afforded
1c in 87% yield.
Ph
Ph
H
1a
1b
1c
2.2. Lipase-catalyzed asymmetric acetylation of 1a
H
H
: C
First, the lipase-catalyzed asymmetric acetylation of racemic 1a
using vinyl acetate as an acyl donor was investigated. In order to
find a suitable enzyme, we screened 19 lipases and some other
enzymes, including proteases and esterases (Table 1). Most of these
enzymes exhibited no or very low catalytic activity for the acetyla-
tion of 1a, although they did have potent catalytic activity toward
general secondary alcohols such as 1-phenylethanol. On the other
hand, all of the lipases that showed low catalytic activity toward
1a also showed extremely high enantioselectivity, affording a single
enantiomer of the acetylated product 3a. The slow reaction rate and
high enantioselectivity in these reactions might be attributed to the
characteristic bulkiness of the carborane. Among the enzymes
examined herein, lipase TL, isolated from Pseudomonas stutzeri,
Ph
unmarked
vertices
: BH
OH
meta-Carborane
1,7-dicarba-closo-
dodecaborane
1d
Figure 1. Structures of carborane-containing secondary alcohols 1a–1d.
The synthetic procedures are shown in Scheme 1. An Ullmann-
type coupling reaction between the C-copper(I) derivative of m-
carborane and iodobenzene gave 1-phenyl-m-carborane 2.
Hydroxyalkyl groups were introduced on the carbon atom of 2
OH
H
H
n-BuLi, CuCl
Ph
H
Ph
n-BuLi
R
Iodobenzene
R-CHO
DME, pyridine
39%
diethyl ether
m-carborane
2
1a
72%
R = Me :
Et : 1b 69%
OH
n-BuLi
n-BuLi
H
Ph
propylene oxide
PhCHO
OH
diethyl ether
28%
diethyl ether
87%
1d
1c
Scheme 1. Synthesis of secondary alcohols 1a–1d.
Table 1
Screening of suitable lipases for the asymmetric acetylation of 1a
OAc
OH
Ph
Ph
vinyl acetate
lipase
i-Pr2O, 50oC
3a
1a
Lipase (origine, supplier)a
AK (Pseudomonas fluorescens, Amano)
AL (Achromobacter sp., Meito)
AYS (Candida rugosa, Meito)
CALB (Candida antarctica, Sigma)
MY (Candida rugosa, Meito)
Time (h) eeS (%)b eeP (%)c Conv. (%)
8
8
8
8
8
8
8
8
8
8
8
17
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
14
3
3
2
2
52
34
0.4
0.4
PL (Alcaligenes sp., Meito)
23
10
19
9
PS (Pseudomonas cepacia, Amano)
QL (Alcaligenes sp., Meito)
SL (Pseudomonas cepacia, Meito)
TL (Pseudomonas stutzeri, Meito)
Type VII (Candida rugosa, Sigma)
77
43
19
50
7
23
> 99
8
a The following enzymes showed no catalytic activity toward 1a: lipase A (Aspergillus sp., Amano),
lipase AS (Aspergillus niger, Amano), lipase F-AP15 (Rhizopus oryzae, Amano), lipase M (Mucor java-
nicus, Amano), lipase OF (Candida rugosa, Meito), lipase R (Penicillium roqueforti, Amano), lipase type II
(porcine pancreas, Sigma), lipase UL (Rhizopus sp., Meito), talipase (Rhizopus delemar, Tanabe), chy-
motrypsin (bovine pancreas, Sigma), newlase F (Rhizopus sp., Amano), papain (Carica papaya, Sigma),
and subtilisin (Bacillus licheniformis, Sigma).
b Enantiomeric excess of the substrate.
c Enantiomeric excess of the product.

S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
1507
Table 2
OH
OH
OAc
Optimization of reaction conditions for the lipase TL-catalyzed asymmetric acetyla-
tion of 1a
Ph
Ph
Ph
vinyl acetate
OAc
OH
+
Ph
Ph
lipase TL
i-Pr2O, 50oC
2.3 h
vinyl acetate
1a
3a
1a
lipase TL
solvent
48%
48%
>99%ee
>99%ee
3a
1a
N-(2-carboxy-4,5-
Temp.
(oC)
30
30
30
30
30
30
30
30
30
40
50
dichlorobenzoyl)-(-)-10,2-
camphorsultam,
Solvent
Time (h)
eeS (%)a
eeP (%)b
Conv. (%)
11
EDC.HCl, DMAP, CH2Cl2
rt, 97%
acetone
DMF
MeCN
DME
i-Pr2O
CH2Cl2
n-hexane
i-octane
i-Pr2O
i-Pr2O
i-Pr2O
i-Pr2O
1
1
1
1
1
1
1
1
0.5
0.5
0.5
2.3
12
0.7
35
2.7
46
3.4
48
30
37
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
0.7
26
2.6
31
3.3
33
23
27
31
36
50
Cl
Cl
N
S
O
O
O
O
O
Ph
44
62
>99
4
50
Scheme 2. Preparation of enantiomerically pure 1a and conversion into ester 4.
a Enantiomeric excess of the substrate.
b Enantiomeric excess of the product.
2.4. Permissiveness of substrate structure
We next investigated the lipase-catalyzed asymmetric acetyla-
tion of 1b, which has an ethyl group at the stereogenic center,
instead of the methyl group of 1a (Table 3). The enantioselectivity
was very high for the acetylation of 1b by lipase TL, as observed in
the reaction of 1a. However, the reaction rate was very low for this
reaction, which proceeded with only 12% conversion in 193 h at
50 °C in diisopropyl ether. Furthermore, acetylation of 1c bearing
a phenyl group did not proceed under the same conditions up to
95 h, although the carboranyl moiety is smaller than that of 1a
due to the lack of a phenyl group on the carborane ring in 1c. Gen-
erally, the lipase-catalyzed acylation of secondary alcohols with a
medium-sized substituent (such as 1-phenylpropanol) at the stere-
ogenic center as the ‘small’ substituent proceeded with a high reac-
tion rate and enantioselectivity, comparable with those of alcohols
with a small-sized substituent, such as 1-phenylethanol.13 In con-
trast to these reports, the permissiveness of the substituent (‘small’
substituent) at the stereogenic center of 1-substituted carboranyl-
methanols is low, probably due to the bulkiness of the spherical
carborane moiety.
In the lipase-catalyzed acylation of substrate 1d with a methy-
lene group between the stereogenic center and carborane moiety,
lipase QL exhibited more efficient catalytic activity than lipase TL
(Table 3). Lipase TL converted only 33% of 1d after 141 h under
the same conditions as those used for 1a, while lipase QL converted
almost all of one enantiomer of 1d into the acetylated product 3d
after 24 h. Although CALB is a useful lipase with a similar range of
substrates to lipase TL, it exhibited a similar reaction rate, but
lower enantioselectivity. This might be due to the longer distance
between the carborane moiety and the stereogenic center in 1d.
exhibited an exceptionally high reaction rate while maintaining
high enantioselectivity: one enantiomer of secondary alcohol 1a
was converted almost completely in 8 h into enantiomerically
pure 3a with an enantiomeric excess of both substrate (ees) and
product (eep) of more than 99%. It has already been reported
that lipase TL acylates relatively bulky substrates, such as benzoin,
with a high reaction rate and enantioselectivity.11
Next, we optimized the reaction conditions for the lipase
TL-catalyzed acetylation of 1a (Table 2). Lipase TL displayed high
catalytic activity in nonpolar organic solvents such as diisopropyl
ether and n-hexane. When diisopropyl ether was used as the sol-
vent, increasing the reaction temperature to 50 °C improved the
reaction rate while maintaining high enantioselectivity. Thus, we
succeeded in the lipase TL-catalyzed kinetic resolution of 1a, with
almost 100% enantiomeric excess of both the product 3a and unre-
acted 1a and a maximum yield of 50% in 2.3 h in diisopropyl ether
at 50 °C.
2.3. Determination of the absolute configuration of the
selectively reacted enantiomer of 1a
HPLC analysis using a chiral column revealed that the same
enantiomer of 1a was selectively acetylated by each usable lipase.
In order to determine the stereochemistry of the catalytic reaction,
the unreacted enantiomer of 1a (ees >99%) after the lipase TL-cat-
alyzed reaction was isolated by silica gel column chromatography,
and was condensed with N-(2-carboxyl-4,5-dichlorobenzoyl)-(À)-
10,2-camphorsultam12 to afford ester 4. X-ray crystallographical
analysis showed that the ester 4 had S-configuration of the carbon
atom attached to the carborane, which indicated that the unre-
acted enantiomer of 1a had an (S)-configuration, and thus that
the lipase selectively acetylates the (R)-enantiomer of 1a
(Scheme 2, Fig. 2). Thus, the lipase recognized the phenylcarborane
moiety as a ‘large’ substituent in the same manner as substituents
such as the phenyl and naphthyl groups of other general secondary
alcohols,8 and selectively acylates the (R)-enantiomer of carbo-
rane-containing secondary alcohols.
2.5. Resolution of a progesterone receptor antagonist candidate
and biological activity of the enantiomers
The application of the lipase-catalyzed enantioselective acyla-
tion of carboranyl alcohols to the resolution of biologically active
compounds was examined next. We had previously proposed that
both C-(cyanophenyl)-m-carborane and C-(cyanophenyl)-p-carbo-

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S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
Figure 2. ORTEP view of 4 synthesized from unreacted 1a in lipase TL-catalyzed reaction, displaying the thermal ellipsoids at 50% probability.
bone structure between 1a and 5. After the isolation of unreacted
(S)-5 and product (R)-7 by silica gel column chromatography,
(R)-7 was hydrolyzed with K2CO3 aq to obtain (R)-5.
Table 3
Permissiveness of substrate structure for the lipase-catalyzed asymmetric acetylation
of carborane-containing secondary alcohols
vinyl acetate
The PR ligand activity of the purified (R)- and (S)-enantiomers of
5 was examined by alkaline phosphatase assay using a T47D
breast-carcinoma cell line (Fig. 3 and Table 4).14 Both enantiomers
of 5 and racemic 5 acted as PR partial agonists, whose maximum
response was approximately 60% of that caused by progesterone.
Compound (S)-5 exhibited more potent agonistic activity than
(R)-5. On the other hand, in the presence of 2 nM progesterone,
compound 5 acted as a PR antagonist at a lower concentration
range compared with its effective concentration as an agonist. Chi-
rality had little influence on the antagonistic activity, compared to
the case of the agonistic activity.
For transcriptional activation of PR, induced conformational
change of the ligand-binding domain (LBD) is important, as is gen-
erally the case for the nuclear receptor family.15 In other words,
potent agonists induce a conformation of the PR LBD that can inter-
act with several transcriptional coactivators, while antagonists
induce slightly or considerably different conformations of the PR
LBD.16 Therefore, activation by an agonist requires a strict confor-
mational change of LBD, while the antagonistic action that resulted
from various LBD conformations differs from the active conforma-
tion, and this may be the reason as to why the chirality of the
ligands has a greater effect on the agonistic activity than on the
antagonistic activity. Thus, enantioseparation using enzymatic
reactions, as illustrated herein, is likely to be useful in medici-
nal–chemical studies of carborane derivatives.
1a 1d
3a 3d
-
-
lipase
i-Pr2O, 50oC
Substrate
Lipase
TL
TL
TL
TL
QL
CALB
Time (h)
2.3
193
eeS (%)a
> 99
eeP (%)b
> 99
> 99
-
> 99
> 99
97
Conv. (%)
1a
1b
1c
1d
1d
1d
50
12
0
33
50
34
14
95
141
24
0
42
> 99
30
141
a Enantiomeric excess of the substrate.
b Enantiomeric excess of the product.
rane bearing a suitable substituent on the other carbon atom of the
carborane would be a key structure for progesterone receptor (PR)
ligands.5 Considering the structure–activity relationships of these
compounds, we designed 1-(7-(4-cyanophenyl)-1,7-dicarba-closo-
dodecaboran-1-yl)ethanol 5 as a novel PR ligand candidate, and
examined the lipase-catalyzed kinetic resolution of racemic 5.
The synthesis and lipase-catalyzed kinetic resolution procedure
of 5 are shown in Scheme 3. Under the optimized reaction condi-
tions for 1a (lipase TL in diisopropyl ether for 2 h at 50 °C), the
asymmetric acetylation of 5 was achieved in maximum yield with
complete enantioresolution (ees, eep >99%). Although the absolute
configurations of unreacted 5 and the acetylated product 7 were
not determined independently, we assume that the selectively
acetylated product was (R)-7 because of the similarity of the back-
3. Conclusion
This is the first report on the enzymatic reaction of boron clus-
ter-containing substrates.17 The present results on the lipase-cata-

S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
1509
NC
NC
OH
H
H
n-BuLi, CuCl
LDA
CH3CHO
H
p-iodobenzonitrile
DME, pyridine
59%
diethyl ether
64%
m-carborane
6
racemic-5
NC
NC
OH
OAc
vinyl acetate
+
lipase TL
i-Pr2O, 50 C
2 h
(S)-5
(R)-7
50% (>99% ee)
48%(>99% ee)
NC
OH
H2O
MeOH
K2CO3
28%
(R)-5
98% ee
Scheme 3. Synthesis and lipase TL-catalyzed resolution of 5.
Table 4
100
80
60
40
20
0
Agonistic and antagonistic activity of both enantiomers of 5 toward progesterone
receptor evaluated by alkaline phosphatase assay using T47D cells
(A)
rac-5
rac-5
R-5
EC50 (µM)a
0.79
IC50 (µM)b
0.037
0.032
0.039
-
Compound
racemic-5
(R)-5
(S)-5
progesterone
ee (%)
R-5
0
S-5
98
1.05
progesterone
PG
> 99
-
0.57
0.0015
a Half-maximum effective concentration of compound as an agonist. The response
of compound 5 was 60% of that induced by progesterone.
b Half-maximum inhibitory concentration of compound 5 toward activity of coex-
isting 2 nM progesterone.
borane as a ‘large substituent’ and selectively acetylate the (R)-
enantiomer in the same manner as general secondary alcohols.
However, the carborane’s bulkiness resulted in a low reaction rate
or poor permissiveness regarding the ‘small’ substituent at the ste-
reogenic center. These results offer useful information about the
scope and limitations of lipase-catalyzed reactions of carborane
derivatives and may also be relevant to other non-natural sub-
strates with bulky substituents. In particular, this methodology
would be effective toward secondary alcohols containing the other
isomers of carborane; o- and p-carboranes. Since carborane is a
useful hydrophobic core structure of ligands for nuclear receptors
and other receptor proteins, the methodology and results reported
herein should also be useful in structure–activity relationship
studies of carborane derivatives.
-10
-9
-8
-7
-6
-5
-4
Log concentration (M)
120
100
80
60
40
20
0
(B)
rraacc-55
RR--55
S-5
S-5
4. Experimental
4.1. General
-10
-9
-8
-7
-6
-5
-4
Log concentration (M)
All reagents were purchased from Sigma-Aldrich, Tokyo Kasei,
Wako Chemicals, and Kanto Chemicals and were used without
further purification. NMR spectra were recorded on Bruker
AVANCE 400 or AVANCE 500 spectrometers. Chemical shifts for
NMR are reported as parts of per million (ppm) relative to
chloroform (7.26 ppm for 1H NMR and 77.16 ppm for 13C NMR).
Figure 3. Dose–response curves of both enantiomers of 5 (A) alone and (B) in the
presence of 2 nM progesterone on alkaline phosphatase activities in T47D cells.
lyzed asymmetric acetylation of secondary alcohols with carborane
at the stereogenic center indicate that some lipases recognize car-

1510
S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
Mass spectra were collected on a Bruker Daltonics microTOF-
2focus spectrometer in the positive and negative ion modes.
HPLC data were recorded with a Hitachi D-2000 Elite type HPLC
1H), 1.75–1.67 (m, 1H), 1.43–1.34 (m, 1H), 1.00 (t, J = 7.4 Hz, 3H);
13C NMR (125 MHz, CDCl3) d 135.4, 128.8, 128.5, 127.9, 82.4,
77.6, 74.4, 30.6, 11.3; HRMS Calcd for
C
11H21B10
O
[MÀH]:
system manager. Chiral column IC (4.6 mm
(4.6 mm
U
 250 mm) and IE
279.2531. Found 279.2532. HPLC: CHIRALPAK IC (25 cm), flow rate
1 mL/min, n-hexane/ethanol = 100/0.3, detection 254 nm, (R)-1b
16.3 min, (S)-1b 12.1 min.
U
 250 mm) were purchased from Daicel Chemical
Industries. All lipase reactions were performed in a Bio Shaker
BR-23 FHÁMR from TAITEC.
4.5. Synthesis of 1-(1,7-dicarba-closo-dodecaboran-1-yl)benzyl
4.2. Synthesis of 1-phenyl-1,7-dicarba-closo-dodecaborane 2
alcohol 1c
At first, 1.6 M n-butyllithium in n-hexane (87.8 mmol) was
added to a solution of m-carborane (11.5 g, 79.8 mmol) in DME
(80 mL) at 0 °C under Ar, and the mixture was stirred at 0 °C for
30 min. Copper(I) chloride (10.3 g, 104 mmol) was added to the
solution at room temperature. After 1 h, pyridine (3.2 mL) and p-
iodobenzene (87.8 mmol) were added, and the mixture was stirred
at 80 °C for 4 h. After cooling, the reaction mixture was diluted
with ether and insoluble materials were filtered off through Celite.
The filtrate was washed with 5% aqueous sodium thiosulfate, 2 M
HCl, and brine, dried over sodium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (eluent:
n-hexane/CH2Cl2, 40/1) to give 2 (39%) as a white solid. 1H NMR
(400 MHz, CDCl3) d 7.44–7.41 (m, 2H), 7.32–7.23 (m, 3H), 3.6–1.6
(br m, 10H), 3.06 (br s, 1H).
At first, 1.6 M n-butyllithium in n-hexane (2.3 mL, 3.8 mmol)
was added to a solution of m-carborane (504 mg, 3.5 mmol) in
diethyl ether (13 mL) at 0 °C under Ar, and the mixture was stirred
at 0 °C for 30 min. Benzaldehyde (0.4 mL, 3.8 mmol) was added to
the solution at 0 °C. The reaction mixture was stirred for 1 h at
room temperature, then poured into saturated ammonium chlo-
ride, and extracted with ether. The organic layer was washed with
water and brine, dried over sodium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (eluent:
n-hexane/ethyl acetate, 10/1) to give 1c (87% yield) as a white
solid. Recrystallization from n-hexane gave plate crystals. Mp
65–66 °C; 1H NMR (500 MHz, CDCl3) d 7.37–7.33 (m, 3H), 7.30–
7.25 (m, 2H), 6.03 (s, 1H), 3.4–1.4 (br m, 10H), 2.86 (br s, 1H);
13C NMR (125 MHz, CDCl3) d 137.7, 129.3, 128.6, 127.4, 78.1,
74.5, 54.8; HRMS Calcd for C9H17B10
O
[MÀH]: 251.2217.
4.3. Synthesis of 1-(7-phenyl-1,7-dicarba-closo-dodecaboran-1-
yl)ethanol 1a
Found 251.2216. HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/
min, n-hexane/ethanol = 100/0.3, detection 254 nm, 12.6 min and
13.3 min (absolute configurations were not identified).
At first, 1.6 M n-butyllithium in n-hexane (7.2 mmol) was added
to a solution of 2 (1.4 g, 6.5 mmol) in diethyl ether (30 mL) at 0 °C
under Ar, and the mixture was stirred at 0 °C for 30 min. Acetalde-
hyde (27 mmol) was added to the solution at 0 °C. The reaction
mixture was stirred for 90 min at room temperature, then poured
into saturated ammonium chloride, and extracted with ether. The
organic layer was washed with water and brine, dried over sodium
sulfate, and concentrated. The residue was purified by silica gel
column chromatography (eluent: n-hexane/ethyl acetate, 8/1) to
give 1a (72% yield) as a white solid. Recrystallization from n-hex-
ane gave plate crystals. Mp 30–31 °C; 1H NMR (400 MHz, CDCl3)
d 7.44–7.42 (m, 2H), 7.30–7.23 (m, 3H), 4.09 (quin, J = 6.4 Hz,
1H), 3.7–1.6 (br m, 10H), 1.90 (d, J = 6.4 Hz, 1H), 1.32 (d,
J = 6.4 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 135.3, 128.8, 128.5,
127.9, 82.5, 77.7, 69.4, 23.8; HRMS Calcd for C10H19B10O [MÀH]:
265.2374. Found 265.2373. HPLC: CHIRALPAK IC (25 cm), flow rate
1 mL/min, n-hexane/ethanol = 100/0.3, detection 254 nm, (R)-1a
16.4 min, (S)-1a 15.3 min. Lipase TL-catalyzed asymmetric acetyla-
tion of rac-1a followed by silica gel column chromatography gave
(S)-1a (>99% ee) as a white solid. Recrystallization from n-hexane
4.6. Synthesis of 1-(7-phenyl-1,7-dicarba-closo-dodecaboran-1-
yl)-2-propanol 1d
At first, 1.6 M n-butyllithium in n-hexane (1.4 mL, 2.2 mmol)
was added to a solution of 2 (452 mg, 2.1 mmol) in diethyl ether
(8 mL) at 0 °C under Ar, and the mixture was stirred at 0 °C for
30 min. Propylene oxide (0.2 mL, 2.5 mmol) was added to the solu-
tion at 0 °C. The reaction mixture was stirred for 1 h at room tem-
perature, then poured into ammonium chloride, and extracted
with ether. The organic layer was washed with water and brine,
dried over sodium sulfate, and concentrated. The residue was puri-
fied by silica gel column chromatography (eluent: n-hexane/ethyl
acetate, 8/1) to give 1d (28% yield) as a white solid. Recrystalliza-
tion from n-hexane gave plate crystals. Mp 69–71 °C; 1H NMR
(400 MHz, CDCl3) d 7.44–7.41 (m, 2H), 7.31–7.25 (m, 3H), 3.90
(m, 1H), 3.6–1.7 (br m, 10H), 2.16 (dd, J = 15.0 and 8.5 Hz, 1H),
2.12 (dd, J = 15.0 and 3.0 Hz, 1H), 1.59 (d J = 4.5 Hz, 1H), 1.17 (d,
J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 135.3, 128.8, 128.5,
127.9, 78.5, 73.7, 67.2, 46.2, 24.1; HRMS Calcd for C11H21B10
O
gave plate crystals. [
a
]
D
25 = À12.3 (c 0.54, CH2Cl2); mp 34–35 °C.
[MÀH]: 279.2531. Found 279.2531. HPLC: CHIRALPAK IC (25 cm),
flow rate 1 mL/min, n-hexane/ethanol = 100/0.3, detection
254 nm, (R)-1d 34.1 min, (S)-1d 12.1 min. Lipase QL-catalyzed
asymmetric acetylation of racemic-1d followed by silica gel column
chromatography gave (S)-1d (estimated configuration, >99% ee) as
4.4. Synthesis of 1-(7-phenyl-1,7-dicarba-closo-dodecaboran-1-
yl)-1-propanol 1b
At first, 1.6 M n-butyllithium in n-hexane (1.5 mL, 2.5 mmol)
was added to a solution of 2 (501 mg, 2.3 mmol) in diethyl ether
(9 mL) at 0 °C under Ar, and the mixture was stirred at 0 °C for
30 min. Propionaldehyde (0.3 mL, 4.3 mmol) was added to the
solution at 0 °C. The reaction mixture was stirred for 1 h at room
temperature, then poured into saturated ammonium chloride,
and extracted with ether. The organic layer was washed with water
and brine, dried over sodium sulfate, and concentrated. The residue
was purified by silica gel column chromatography (eluent: n-hex-
ane/ethyl acetate, 5/1) to give 1b (69% yield) as a white solid.
Recrystallization from n-hexane gave plate crystals. Mp 66–
67 °C; 1H NMR (400 MHz, CDCl3) d 7.44–7.41 (m, 2H), 7.29–7.25
(m, 3H), 3.73 (m, 1H), 3.6–1.6 (br m, 10H), 1.84 (d, J = 6.4 Hz,
a colorless liquid. [a]
25 = +23.5 (c 0.3, CH2Cl2).
D
4.7. Synthesis of 4
N-(2-Carboxy-4,5-dichlorobenzoyl)-(À)-10,2-camphorsultam
(112 mg, 0.26 mmol) was added to a solution of 1a (recovered from
the enzymatic reaction mixture, Scheme 2, 44 mg, 0.17 mmol) in
dichloromethane (5 mL) at room temperature. Next, EDCÁHCl
(42 mg, 0.2 mmol) and DMAP (4.2 mg, 0.03 mmol) were added.
The reaction mixture was stirred for 17 h at room temperature
and then poured into water. The organic layer was washed with
2 M hydrochloric acid, saturated sodium bicarbonate, and brine,
dried over sodium sulfate, and concentrated. The crude product

S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
1511
was purified by recrystallization from DMF to give 4 (97% yield) as
4.10.1. 1-(7-Phenyl-1,7-dicarba-closo-dodecaboran-1-yl)ethyl
needles. [
a]
25 = À82.1 (c 0.2, CH2Cl2); mp 164–166 °C; 1H NMR
acetate 3a
D
(500 MHz, CDCl3) d 8.05 (s, 1H), 7.54 (s, 1H), 7.43–7.40 (m, 2H),
7.31–7.21 (m, 3H), 5.40 (q, J = 6.5 Hz, 1H), 4.05 (br s, 1H), 3.6–1.5
(br m, 10H), 3.42 (d, J = 13.5 Hz, 1H), 3.38 (d, J = 13.5 Hz, 1H),
2.47 (br d, J = 13.0 Hz, 1H), 2.18 (m, 1H), 1.98–1.88 (m, 3H),
1.46–1.38 (m, 2H), 1.38 (d, J = 6.5 Hz, 3H), 1.16 (s, 3H), 0.97 (s,
3H); 13C NMR (125 MHz, CDCl3) d 165.2, 161.8, 137.6 135.3,
135.3, 134.9, 131.6, 131.2, 129.0, 128.6, 128.0, 127.9, 77.9, 77.8,
71.0, 65.9, 53.3, 48.6, 47.9, 44.9, 37.6, 33.3, 26.6, 21.0, 20.7, 20.2;
HRMS Calcd for C28H37B10Cl2NO5SNa [M+Na]: 702.2636. Found
702.2636.
Yield: 83%; colorless liquid; 1H NMR (500 MHz, CDCl3) d 7.43–
7.40 (m, 2H), 7.32–7.24 (m, 3H), 5.27 (q, J = 6.5 Hz, 1H), 3.7–1.5
(br m, 10H), 2.08 (s, 3H), 1.30 (d, J = 6.5 Hz, 3H); 13C NMR
(125 MHz, CDCl3) d 169.4, 135.2, 128.9, 128.5, 127.9, 78.5, 77.6,
69.3, 21.3, 21.1; HRMS Calcd for
C12H22B10O2Na [M+Na]:
331.2445. Found 331.2439. HPLC: CHIRALPAK IC (25 cm), flow rate
1 mL/min, n-hexane/ethanol = 100/0.3, detection 254 nm, (R)-3a
5.4 min, (S)-3a 4.8 min. Lipase TL-catalyzed asymmetric acetyla-
tion of rac-1a followed by silica gel column chromatography gave
(R)-3a (>99% ee) as a colorless liquid. [a]
25 = +68.0 (c 0.42, CH2Cl2).
D
4.8. Synthesis of 1-(4-cyanophenyl)-1,7-dicarba-closo-dodecab-
orane 6
4.10.2. 1-(7-Phenyl-1,7-dicarba-closo-dodecaboran-1-yl)-
propan-1-yl acetate 3b
Yield: 70%; colorless liquid; 1H NMR (400 MHz, CDCl3) d 7.41–
7.39 (m, 2H), 7.29–7.25 (m, 3H), 5.17 (dd, J = 10.8 and
2.8 Hz, 1H), 3.7–1.5 (br m, 10H), 2.11 (s, 3H), 1.78–1.68 (m, 1H),
1.61–1.49 (m, 1H), 0.87 (t, J = 7.6 Hz, 3H); 13C NMR (125 MHz,
CDCl3) d 169.9, 135.2, 128.9, 78.5, 77.6, 73.1, 28.8, 20.8, 10.7;
At first, 1.6 M n-butyllithium, in n-hexane (7.4 mL, 11.9 mmol)
was added to a solution of m-carborane (1.5 g, 10.8 mmol) in
DME (12 mL) at 0 °C under Ar, and the mixture was stirred at
0 °C for 30 min. Copper(I) chloride (10.3 mg, 104 mmol) was added
to the solution at room temperature. After 1 h, pyridine (3.8 mL)
and p-iodobenzonitrile (2.8 g, 11.9 mmol) were added, and the
mixture was stirred at 80 °C for 2.5 h. After cooling, the reaction
mixture was diluted with ether, and insoluble materials were fil-
tered off through Celite. The filtrate was washed with 5% sodium
thiosulfate, 2 M hydrochloric acid, and brine, dried over sodium
sulfate, and then concentrated. The residue was purified by silica
gel column chromatography (eluent: n-hexane/ethyl acetate, 20/
1) to give 6 (59% yield) as a white solid. 1H NMR (400 MHz, CDCl3)
d 7.58–7.53 (m, 4H), 3.5–1.7 (br m, 10H), 3.12 (br s, 1H).
HRMS Calcd for
C13H24B10O2Na [M+Na]: 345.2602. Found
345.2602. HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hex-
ane/ethanol = 100/0.3, detection 254 nm, R-3b 5.4 min, S-3b
4.9 min. Lipase TL-catalyzed asymmetric acetylation of rac-1b fol-
lowed by silica gel column chromatography gave R-3b (estimated
configuration, >99% ee) as a colorless liquid. [
CH2Cl2).
a]
25 = +52.9 (c 0.22,
D
4.10.3. 1-(1,7-Dicarba-closo-dodecaboran-1-yl)benzyl acetate 3c
Yield: 97%; recrystallization from n-hexane gave plate crystals.
Mp 64–65 °C; 1H NMR (400 MHz, CDCl3) d 7.34–7.33 (m, 2H), 7.28–
7.23 (m, 3H), 6.03 (s, 1H), 3.5–1.5 (br m, 10H), 2.86 (br s, 1H), 2.17
(s, 3H); 13C NMR (125 MHz, CDCl3) d 135.3, 128.8, 128.5, 127.9,
69.4, 23.8; HRMS Calcd for C11H20B10O2Na [M+Na]: 345.2602.
Found 345.2602. HPLC: CHIRALPAK IC (25 cm), flow rate 1 mL/
min, n-hexane/ethanol = 100/0.3, detection 254 nm, 5.5 min and
6.1 min (absolute configurations were not identified).
4.9. Synthesis of 1-(7-(4-cyanophenyl)-1,7-dicarba-closo-dodec-
aboran-1-yl)ethanol 5
At first, 1.1 M lithium diisopropylamide in n-hexane-tetrahy-
drofuran (8.7 mL, 9.7 mmol) was added to a solution of 6 (2.0 g,
8.1 mmol) in THF (30 mL) at À78 °C under Ar, and the mixture
was stirred at À78 °C for 10 min. Acetaldehyde (9.0 mL) was then
added to the solution at À78 °C. The reaction mixture was stirred
for 30 min at À78 °C, then poured into saturated ammonium chlo-
ride, and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over sodium sulfate, and con-
centrated. The residue was purified by silica gel column chroma-
tography (eluent: n-hexane/ethyl acetate, 8/1) to give 5 (64%
yield) as a white solid. Mp 82–83 °C; 1H NMR (500 MHz, CDCl3) d
7.58–7.53 (m, 4H), 4.09 (quin, J = 6.0 Hz, 1H), 3.7–1.8 (br m,
10H), 1.92 (d, J = 6.0 Hz, 1H), 1.32 (d, J = 6.0 Hz, 3H); 13C NMR
(500 MHz, CDCl3) d 140.0, 132.3, 128.8, 118.1, 112.9, 82.9, 75.8,
69.3, 23.8; Anal. Calcd for: C11B10H19NO: C, 45.65; H, 6.62; N,
4.84. Found: C, 45.35; H, 6.53; N, 4.83; HRMS Calcd for C11H18B10-
NO [MÀH]: 290.2327. Found 290.2327. HPLC: CHIRALPAK IE
(25 cm), flow rate 1 mL/min, n-hexane/ethanol = 90/10, detection
254 nm, (R)-5 10.0 min, (S)-5 8.1 min.
4.10.4. 1-(7-Phenyl-1,7-dicarba-closo-dodecaboran-1-yl)-
propan-2-yl acetate 3d
Yield: 80%; recrystallization from n-hexane gave plate crystals.
Mp 49–50 °C; 1H NMR (500 MHz, CDCl3) d 7.42–7.39 (m, 2H), 7.31–
7.23 (m, 3H), 4.85 (m, 1H), 3.6–1.7 (br m, 10H), 2.38 (dd, J = 15.5
and 10.0 Hz, 1H), 2.13 (dd, J = 15.5 and 2.5 Hz, 1H), 2.05 (s, 1H),
1.19 (d, J = 6.5 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 170.5, 135.3,
128.8, 128.5, 127.9, 78.4, 73.0, 70.1, 42.8, 21.5, 20.6; HRMS Calcd
for C13H24B10O2Na [M+Na]: 345.2602. Found 345.2606. HPLC:
CHIRALPAK IC (25 cm), flow rate 1 mL/min, n-hexane/etha-
nol = 100/0.3, detection 254 nm, (R)-3d 6.5 min, (S)-3d 6.9 min.
Lipase QL-catalyzed asymmetric acetylation of rac-1d followed
by silica gel column chromatography gave (R)-3d (estimated con-
figuration, >99% ee) as a white solid. Recrystallization from n-hex-
ane gave plate crystals. Mp 64–65 °C; specific rotation was not
detected at (c 0.4, CH2Cl2).
4.10. General procedure for the synthesis of acetylated products
of the lipase-catalyzed reaction for their use as authentic
samples in HPLC
4.10.5. 1-(7-(4-Cyanophenyl)-1,7-dicarba-closo-dodecaboran-1-
yl)ethyl acetate 7
Yield: 92%; colorless liquid; 1H NMR (400 MHz, CDCl3) d 7.57 (d,
J = 10.8 Hz, 2H), 7.52 (d, J = 10.8 Hz, 2H), 5.27 (q, J = 6.4 Hz, 1H),
3.5–1.6 (br m, 10H), 2.09 (s, 3H), 1.30 (d, J = 6.8 Hz, 3H); 13C NMR
(125 MHz, CDCl3) d 169.9, 140.0, 132.3, 128.8, 118.1, 112.9, 82.9,
75.8, 70.2, 23.8, 22.3; HRMS Calcd for C13H21B10NO2Na [M+Na]:
345.2398. Found 356.2397. HPLC: CHIRALPAK IC (25 cm), flow rate
1 mL/min, n-hexane/ethanol = 90/10, detection 254 nm, R-7
7.5 min, S-7 7.2 min.
Triethylamine (162 mg, 1.6 mmol) and acetic anhydride (98 mg,
1.0 mmol) were added to a solution of alcohol (0.3 mmol) in
dichloromethane (2.4 mL). The reaction mixture was stirred for
72 h at room temperature and then poured into 2 M hydrochloric
acid. The organic layer was washed with water and brine, dried
over sodium sulfate, and concentrated. The residue was purified
by silica gel column chromatography to give the acetylated
product.

1512
S. Mori et al. / Tetrahedron: Asymmetry 25 (2014) 1505–1512
4.11. General procedure for the lipase-catalyzed asymmetric
acetylation of alcohols
Acknowledgments
This work was partly supported by JSPS KAKENHI Grant Nos.
23790128 and 25360146 (to F.S.) and No. 22136013 (to H.K.), JSPS
Core-to-Core Program, A. Advanced Research Networks, and MEXT
Platform for Drug Discovery, Informatics, and Structural Life
Science, Japan.
Lipase (15 mg) was added to a vial containing 1 mL of a solution
of the racemic alcohol (0.02 mmol) and vinyl acetate (0.2 mmol),
and the reaction mixture was shaken at 120 rounds/min at con-
stant temperature. At frequent intervals, aliquots were withdrawn
and filtered with a syringe filter, followed by HPLC analysis.
References
4.12. Preparation of the (R)- and (S)-enantiomers of 5 by lipase
TL-catalyzed asymmetric acetylation
1. For reviews of carboranes: (a) Bregadze, V. I. Chem. Rev. 1992, 92, 209; (b)
Hawthorne, M. F. Angew. Chem., Int. Ed. 1993, 32, 950; (c) Soloway, A. H.; Tjarks,
W.; Barnum, J. G. Chem. Rev. 1998, 98, 1515.
2. (a) Iijima, T.; Endo, Y.; Tsuji, M.; Kawachi, E.; Kagechika, H.; Shudo, K. Chem.
Pharm. Bull. 1999, 47, 398; (b) Ohta, K.; Iijima, T.; Kawachi, E.; Kagechika, H.;
Endo, Y. Bioorg. Med. Chem. Lett. 2004, 14, 5913.
3. (a) Endo, Y.; Iijima, T.; Yamakoshi, Y.; Yamaguchi, M.; Fukasawa, H.; Shudo, K. J.
Med. Chem. 1999, 42, 1501; (b) Endo, Y.; Iijima, T.; Yamakoshi, Y.; Fukasawa, H.;
Miyaura, C.; Inada, M.; Kubo, A.; Itai, A. Chem. Biol. 2001, 8, 341; (c) Endo, Y.;
Yoshimi, T.; Ohta, K.; Suzuki, T.; Ohta, S. J. Med. Chem. 2005, 48, 3941.
4. (a) Fujii, S.; Yamada, A.; Tomita, K.; Nagano, M.; Goto, T.; Ohta, K.; Harayama,
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K.; Fujii, S.; Ohta, S.; Endo, Y. J. Med. Chem. 2010, 53, 4917; (c) Fujii, S.; Goto, T.;
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4654; (d) Fujii, S.; Hashimoto, Y.; Suzuki, T.; Ohta, S.; Endo, Y. Bioorg. Med.
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5. (a) Fujii, S.; Yamada, A.; Nakano, E.; Takeuchi, Y.; Mori, S.; Masuno, H.;
Kagechika, H. Eur. J. Med. Chem. 2014, 84, 264; (b) Fujii, S.; Nakano, E.;
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6. (a) Fujii, S.; Masuno, H.; Taoda, Y.; Kano, A.; Wongmayura, A.; Nakabayashi, M.;
Ito, N.; Shimizu, M.; Kawachi, E.; Hirano, T.; Endo, Y.; Tanatani, A.; Kagechika,
H. J. Am. Chem. Soc. 2011, 133, 20933; (b) Fujii, S.; Kano, A.; Masuno, H.;
Songkram, C.; Kawachi, E.; Hirano, T.; Tanatani, A.; Kageshika, H. Bioorg. Med.
Chem. Lett. 2014, 24, 4515.
7. For reviews of lipase: (a) Halling, P. J. Curr. Opin. Biotechnol. 2000, 4, 74; (b)
Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39, 2226; (c) Klibanov, A. M.
Nature 2001, 409, 241; (d) Berglund, P. Biomol. Eng. 2001, 18, 13; (e) Bordusa, F.
Chem. Rev. 2002, 102, 4817; (f) Ghanem, A.; Aboul-Enein, H. Y. Tetrahedron:
Asymmetry 2004, 15, 3331; (g) Ema, T. Curr. Org. Chem. 2004, 8, 1009; (h)
Ghanem, A.; Enein, A. Chirality 2005, 17, 1.
8. (a) Kazlauskas, A. N. E.; Weiissfloch, A. T.; Cuccia, J. J. Org. Chem. 1991, 56,
2656; (b) Ema, T.; Yamaguchi, K.; Wakasa, Y.; Yabe, A.; Okada, R.; Fukumoto,
M.; Yano, F.; Korenaga, T.; Utaka, M.; Sakai, T. J. Mol. Cat. B: Enzym. 2003, 22,
181.
Lipase TL (1 g) was added to a solution of vinyl acetate
(21 mmol) and racemic 5 (600 mg, 2.1 mmol) in diisopropyl ether
(20 mL) and the mixture was stirred at 50 °C for 2 h. After removal
of the lipase by filtration, the filtrate was concentrated. The residue
was purified by silica gel column chromatography (eluent: n-hex-
ane/ethyl acetate, 10/1) to give (S)-5 (50% yield, >99% ee) as a col-
orless solid and (R)-7 (49% yield, >99% ee) as a colorless liquid.
Next, (R)-7 was dissolved in 0.43 M potassium carbonate in meth-
anol/water (4:1) and the mixture was stirred for 40 min at room
temperature. The reaction mixture was extracted with ethyl ace-
tate, dried over sodium sulfate, and then concentrated. The residue
was purified by silica gel column chromatography (eluent: n-hex-
ane/ethyl acetate, 4/1) to give (R)-5 (28% yield, 98% ee) as a color-
less solid. Recrystallization of (R)- and (S)-5 from n-hexane gave
plate crystals. (R)-5: [a]
25 = +10.2 (c 0.5, CH2Cl2); mp 98–99 °C;
D
Anal. Calcd for: C11B10H19NO: C, 45.65; H, 6.62; N, 4.84. Found: C,
45.60; H, 6.75; N, 4.83; S-5: [
100 °C; Anal. Calcd for: C11B10H19NO: C, 45.65; H, 6.62; N, 4.84.
a
]
25 = À10.8 (c 1.0, CH2Cl2); mp 99–
D
Found: C, 45.80; H, 6.51; N, 4.56.
4.13. X-ray crystallography
Crystallographic data of compound 4 were collected on a Bruker
ApexII CCD detector with graphite-monochromated Mo K
Quantum210. Crystal system: monoclinic; space group: P21; unit
cell dimensions: a = 20.92 (7) Å, b = 7.460 (13) Å, c = 23.51 (4) Å,
B = 112.9 (3)°, final R indices [I > 2r(I)] R1 = 0.0707, wR2 = 0.1576.
The crystallographic data for 4 have been deposited at the
Cambridge Crystallographic Data Centre as CCDC 1031421.
a and
9. Janssen, A. J. M.; Klunder, A. J. H.; Zwanenburg, B. Tetrahedron 1991, 47, 7645.
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12. Harada, N.; Koumura, N.; Robillard, M. Enantiomer 1997, 2, 303.
13. For example see: (a) Negi, S.; Umetsu, K.; Nishijo, Y.; Kano, K.; Nakamura, K.
Enantiomer 2000, 5, 63; (b) Nakamura, K.; Murata, M.; Tanaka, R.; Yano, M.;
Hirose, K.; Tobe, Y. Tetrahedron: Asymmetry 1996, 7, 3285; (c) Córdova, A.;
Tremblay, M. R.; Clapham, B.; Janda, K. D. J. Org. Chem. 2001, 66, 5645.
14. Lorenzo, D. D.; Albertini, A.; Zava, D. Cancer Res. 1991, 51, 4470.
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16. (a) Raaijmakers, H. A.; Versteegh, J. E.; Uitdehaag, J. M. J. Biol. Chem. 2009, 284,
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Bray, J. D. Mol. Endocrinol. 2007, 21, 1066.
4.14. Alkaline phosphatase assay using T47D cells
T47D breast-carcinoma cells were cultured in an RPMI 1640
medium with 10% (v/v) fetal bovine serum. Cells were plated in
96-well plates at 104 cells/well and incubated overnight (37 °C,
5% CO2 in air). The next day, cells were treated with fresh medium
containing test compound, and further incubated for 24 h. The
medium was aspirated and the cells were fixed with 100
1.8% formalin (in PBS). The fixed cells were washed with PBS and
75 L of assay buffer (1 mg/mL p-nitrophenol phosphate in dieth-
lL of
17. Some enzymatic reactions toward boronic acid or boronate ester-containing
substrates have been reported: (a) Andrade, L. H.; Barcellos, T. Org. Lett. 2009,
11, 3052; (b) Kim, C.; Lee, J.; Cho, J.; Oh, Y.; Choi, Y. K.; Choi, E.; Park, J.; Kim, M.-
J. J. Org. Chem. 2013, 78, 2571.
l
anolamine water solution, pH 9.0, 2 mM MgCl2) was added. The
mixture was incubated at room temperature with shielding from
light for 2 h, and then the reaction was terminated by the addition
of 100 lL of NaOH. The absorbance at 405 nm was measured.