In situ evaluation of kinetic resolution catalysts for nitroaldol by rationally designed fluorescence probe

Article abstract of DOI:10.1021/jo1020344

Development of effective chemical catalysts is a key concern in organic chemistry. Therefore, convenient screening systems for chemical catalysts are required, and although some fluorescence-based HTS systems have been developed, little attempt has been made to apply them to asymmetric catalysts. Therefore, we tried to develop a chiral fluorescence probe which can evaluate the reactivity and enantioselectivity of asymmetric catalysts. We focused on kinetic resolution catalysts as a target of our novel fluorescence probe, employing β-elimination following acylation of nitroaldol. Once the hydroxyl group of nitroaldol is acylated, β-elimination occurs immediately, affording nitro olefin. Therefore, we designed and synthesized a fluorescence probe with an asymmetric nitroaldol moiety. Its fluorescence intensity decreases dramatically upon β-elimination, so the fluorescence decrease is an indicator of the reaction yield. Thus, the enantioselectivity of kinetic resolution catalysts can be assessed simply by measuring the fluorescence intensities of the reaction mixtures of the two enantiomers; it is not necessary to purify the product. This fluorescence probe revealed that benzotetramisole is a superior catalyst for kinetic resolution of nitroaldol. Furthermore, we established an HTS system for asymmetric catalysts, using a fluorescence probe and benzotetramisole. To our knowledge, this is the first fluorescence-based HTS system for asymmetric catalysts.

Full text of DOI:10.1021/jo1020344

FEATURED ARTICLE
pubs.acs.org/joc
In Situ Evaluation of Kinetic Resolution Catalysts for Nitroaldol
by Rationally Designed Fluorescence Probe
||
||
||
Takuya Matsumoto,†, Yasuteru Urano,† Yoshinori Takahashi,‡, Yusuke Mori,‡,§,|| Takuya Terai,†, and
Tetsuo Nagano*,†,
||
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
§SOSHO, Inc., Osaka 541-0053, Japan
CREST, JST, Kawaguchi, Saitama 332-0012, Japan
S Supporting Information
b
ABSTRACT: Development of effective chemical catalysts is a
key concern in organic chemistry. Therefore, convenient
screening systems for chemical catalysts are required, and
although some fluorescence-based HTS systems have been
developed, little attempt has been made to apply them to
asymmetric catalysts. Therefore, we tried to develop a chiral
fluorescence probe which can evaluate the reactivity and
enantioselectivity of asymmetric catalysts. We focused on kinetic resolution catalysts as a target of our novel fluorescence probe,
employing β-elimination following acylation of nitroaldol. Once the hydroxyl group of nitroaldol is acylated, β-elimination occurs
immediately, affording nitro olefin. Therefore, we designed and synthesized a fluorescence probe with an asymmetric nitroaldol
moiety. Its fluorescence intensity decreases dramatically upon β-elimination, so the fluorescence decrease is an indicator of the
reaction yield. Thus, the enantioselectivity of kinetic resolution catalysts can be assessed simply by measuring the fluorescence
intensities of the reaction mixtures of the two enantiomers; it is not necessary to purify the product. This fluorescence probe revealed
that benzotetramisole is a superior catalyst for kinetic resolution of nitroaldol. Furthermore, we established an HTS system for
asymmetric catalysts, using a fluorescence probe and benzotetramisole. To our knowledge, this is the first fluorescence-based HTS
system for asymmetric catalysts.
’ INTRODUCTION
have attempted to develop HTS for chemical catalysts by means
of MS,4,5 NMR,6 CD,7-10 thermography,11 fluorescence,12-29
and so on. Among these techniques, fluorescence-based HTS
seems the most useful, because fluorescence is easily measured,
even if the solution contains impurities, and purification is
unnecessary. But, most conventional fluorescence HTS systems
focus on determining not enantioselectivity, but reaction yield.
Therefore, to develop fluorescence-based HTS system for asym-
metric catalysts is both important and challenging. For this
reason, we tried to develop a novel chiral fluorescence probe
whose fluorescence is controlled by asymmetric chemical
reaction.
As a target of HTS we focused on the Henry reaction, which is
a fundamental reaction for carbon-carbon bond formation and
yields nitroaldol. Nitroaldol is a very important compound in
pharmaceutical and synthetic chemistry because it is easily
transformed into amino alcohol, nitro ketone, nitro olefin, and
so on.30-37 Nitroaldol often contains an asymmetric carbon, so
we tried to develop an HTS system for kinetic resolution catalysts
of nitroaldol. Here we report a rationally designed chiral
Fluorescence probes are powerful tools in many fields of
chemistry and biology, and have recently been introduced for
high-throughput screening (HTS) of chemical catalysts. The
creation of effective chemical catalysts is a central topic in organic
chemistry, and although some have been designed on the basis of
reaction mechanism, most have been developed through trial and
error. Nowadays, combinatorial chemistry is used to obtain large
libraries of candidate catalysts, but the yield and enantiomeric
excess (ee) of the products are still determined by using high-
performance liquid chromatography (HPLC) and gas chroma-
tography (GC), which represent a bottleneck due to the rela-
tively long analysis time. Optimizing the reaction conditions, i.e.,
solvent, concentration, temperature, time, etc., is also time-
consuming. Therefore, a convenient HTS system for evaluation
of the activities and enantioselectivities of many candidate
catalysts in parallel would be very useful.1-3
HTS is already indispensable for discovering candidate drugs
and for optimizing lead structures in pharmaceutical chemistry.
Fluorescence detection is most commonly used in HTS systems,
because its sensitivity is especially high, and in the case of
screening for catalysts, it can be used to quantify the chemical
yields in situ without purification of products. Several groups
Received: October 13, 2010
Published: March 03, 2011
r
2011 American Chemical Society
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FEATURED ARTICLE
fluorescence probe suitable for an HTS system aimed at kinetic
resolution catalysts of nitroaldol, using BODIPY as a fluoro-
phore.
can lead to a fluorescence increase, and rational molecular design
would allow us to maximize the fluorescence change caused by
the reaction. We therefore employed BODIPY and PET to create
a novel fluorescence probe which is strongly fluorescent before
reaction (Figure 1b), but almost nonfluorescent (Figure 1c) after
β-elimination, as a tool for an HTS system to find kinetic
resolution catalysts of nitroaldol.
As shown in Scheme 1, once the hydroxyl group of a nitroaldol
is acylated, β-elimination occurs immediately, and nitro olefin is
produced. Nitro olefin is an electron acceptor for the PET
process, and quenches the fluorescence. If β-elimination occurs,
we thought that the progress of the acylation could be evaluated
simply by measuring the fluorescence decrease of the reaction
mixture. The difference between the changes of fluorescence
intensities of the two enantiomers reflects the enantioselectivity
of a kinetic resolution catalyst.
To test our hypothesis, these compounds were synthesized
and their fluorescence properties were measured. Synthesis was
performed according to Scheme 2. Nitroaldol BODIPY (6) was
synthesized in 6 steps. When the hydroxyl group of 6 was
acylated, nitro olefin BODIPY (8) was produced immediately,
as we had expected. Next, the fluorescence spectra and properties
of 6 and 8 were examined. The fluorescence and absorbance
spectra are illustrated in Figure 2. As shown in Figure 2, the
absorbance spectrum was not changed after elimination, but the
fluorescence was dramatically decreased. Thus, nitro olefin
appeared to act as an electron acceptor for PET, and the
fluorescence of BODIPY was quenched. The decrease of fluor-
escence quantum yield (Φfl) also indicated that nitro olefin could
quench the fluorescence of BODIPY; Φfl of 8 is extremely low
(see Supporting Information).
’ RESULTS AND DISCUSSION
Development of HTS System for Kinetic Resolution Cata-
lysts. BODIPY has been used not only as a fluorescent tag, but
also as a core of fluorescence probes, because of its high fluo-
rescence quantum yield with excitation in the visible range, and
its relatively strong resistance to photobleaching. Furthermore,
the fluorescence is not influenced by solvents and is relatively
stable against chemical reaction.38-40 These features are all
favorable for application to HTS of chemical catalysts. Our group
has designed and developed many BODIPY fluorescence probes
based on photoinduced electron transfer (PET).41-44 PET is a
comprehensively reported mechanism for excited-state quench-
ing, in which an electron is transferred from an electron donor
moiety to an electron acceptor moiety (Figure 1a). If the electron
acceptor has a low LUMO energy level, PET occurs and the
fluorescence of the electron donor is quenched (Figure 1c).45,46
A fluorescence probe controlled by PET would have the advan-
tage that structural change at a moiety other than the fluorophore
The fluorescence change upon elimination was thus as ex-
pected, and 6 was found to be a superior fluorescence probe,
which was expected to be suitable for use in an HTS system for
kinetic resolution catalysts.
The strategy for an HTS system for kinetic resolution catalysts
is illustrated in Figure 3. For example, if a candidate compound
could catalyze acylation of (R)-6 but could not catalyze acylation
of (S)-6, the fluorescence intensity due to (R)-6 would decrease
but that due to (S)-6 would not. So, the difference of fluorescence
intensity reflects the enantioselectivity of the catalyst.
Before developing an HTS system for kinetic resolution cata-
lysts, we performed optical resolution of racemic 6 by means of
HPLC with a CHIRALPAK IA (DAICEL) column and 2-propa-
nol/n-hexane=1/9astheeluent(seetheSupportingInformation).
The separated enantiomers of 6 were iodinated according to
Scheme 3 because a heavy atom was needed for accurate assign-
ment of the absolute configuration. Iodinated (R)-9 was recrys-
tallized from CH2Cl2/n-hexane under stirring with a rotary
shaker47,48 at the rotation rate of 50 rpm for X-ray crystallography.
The ORTEP plot of (R)-9 is shown in the Supporting Information.
Figure 1. Schematic representation of PET strategy to control fluores-
cence: (a) switch “off” model in which PET occurs, resulting no
fluorescence; (b), (c) schematic representation of relationship between
LUMO energy level and PET; (b) when the electron acceptor has a high
LUMO energy level, PET does not occur and fluorescence is not
quenched; (c) when the electron acceptor has a low LUMO energy
level, PET occurs and fluorescence is quenched.
Scheme 1. Acylation and Elimination of Nitroaldol
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FEATURED ARTICLE
Scheme 2. Synthetic Scheme of Nitroaldol BODIPY(6) and Nitro Olefin BODIPY(8)a
a Reagents: (a) NaBH4, THF, 25%; (b) acetic anhydride, pyridine, 67%; (c) (1) 2,4-dimethylpyrrole, TFA, CH2Cl2, (2) DDQ, (3) DIEA, BF3 Et2O,
3
toluene, 26%; (d) NaOH aq, MeOH/CH2Cl2, quant; (e) PCC, CH2Cl2, 68%; (f) CH3NO2, NaOH aq, MeOH, 93%; (g) Ac2O, pyridine, CH2Cl2, 86%.
With our novel HTS tool for kinetic resolution catalysts, we
tested the enantioselectivity of some commercially available
catalysts which have been used for acylation of chiral secondary
alcohols. Ferrocene derivative49 ((R)-10), histidine derivative50
((S)-11), and benzotetramisole51-53 ((R)-12) were used as
catalysts, and propionic anhydride, butyric anhydride, and iso-
butyric anhydride were used as acylating reagents. Acylation was
performed in DMF, MeCN, or toluene at room temperature for
1 h, and a small portion of each reaction mixture was sampled and
diluted 100 times with DMSO to 10 μM for fluorescence
measurement. In the fluorescence assay, the fluorescence inten-
sity cannot be measured at too high a dye concentration because
self-quenching of the dye occurs; in the present case, 10 μM was
appropriate, affording a linear calibration curve. The reaction
yield in each case was obtained from the calibration curve by
interpolation of the observed fluorescence intensity (see the
Experimental Section and Supporting Information). The sub-
strate 6 is strongly fluorescent, whereas product 8 is not
fluorescent, so high fluorescence intensity corresponds to low
reaction yield and low fluorescence intensity corresponds to high
reaction yield.
substitute alcohol.54 Thus, the fact that the bulky acylating
reagent has good enantioselectivity is reasonable. To confirm the
validity of our HTS system, we evaluated the results in these cases
by HPLC analysis. For HPLC analysis, racemic 6 was treated
with (R)-12 and each acid anhydride in toluene, and after the
reaction, the yield and % ee were determined by means of HPLC
in a chiral column. The selectivity was estimated as follows.55,56
kfast
ln½ð1 - cÞð1 - eeÞꢀ
ln½ð1 - cÞð1 þ eeÞꢀ
s ¼
¼
kslow
Calculated selectivity values are summarized in Table 1. As in
the case of fluorescence HTS, isobutyric anhydride had the best
selectivity value among the three acid anhydrides. Because this
HPLC assay and our fluorescence assay provided the same result,
our fluorescence HTS system appears to be suitable to evaluate
the activity and enantioselectivity of kinetic resolution catalysts.
Moreover, a major advantage of the fluorescence probe is that
this system can monitor the chemical reaction in situ, so we next
examined real-time reaction monitoring with our probe. As
shown in Figure 5, the reaction was performed in a fluorescence
spectrometer and the fluorescence intensity of the reaction
mixture was measured every 0.5 s. (R)- or (S)-6 was dehydrated
by propionic anhydride, butyric anhydride, or isobutyric anhy-
dride in toluene in the presence of (R)-12 as a catalyst. When
propionic anhydride was used, the reaction was the fastest, but
the enantioselectivity was low. When isobutyric anhydride was
As shown in Figure 4, when (R)-12 was used as a catalyst
in toluene, isobutyric anhydride was the best acylating reagent,
and propionic anhydride and butyric anhydride gave lower
enantioselectivity. Enantioselectivity of benzotetramisole is due
to π-π interaction between acylated benzotetramisole and
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Figure 2. Fluorescence and absorbance spectra of 6 and 8. The spectra of 1 μM dyes were measured in DMSO.
Scheme 3. Iodination of (R)-6a
a Reagents: (a) I2, HIO3, EtOH/H2O/CH2Cl2, 84%.
fluorescence-based assay revealed that (R)-12 is a good kinetic
resolution catalyst for nitroaldol, and the Henry reaction is
widely used to obtain nitroaldol. Therefore, we aimed to establish
an HTS system to estimate the reaction yield and % ee based on
the fluorescence decrease resulting from reaction of 6 to afford 8,
as shown in Scheme 4. In order to determine the reactivity and
enantioselectivity of a catalyst that catalyzes the first Henry
reaction step, we need to measure the amounts of (R)-6 and
(S)-6. We thought that these amounts could be obtained from
the fluorescence decrease in the second % ee determination step,
similar to the enzymatic method for determining enantiomeric
excess (EMDee).
Figure 3. Schematic illustration of strategy of HTS system for kinetic
resolution catalysts.
used, the reaction was the slowest, while enantioselectivity was
high. This tendency is the same as in Table 1, confirming the
usefulness of our fluorescence probe for kinetic study (see the
Supporting Information).
HTS System for % ee Determination. Next, we tried to
establish an HTS system to evaluate the reactivity and enantios-
electivity of asymmetric catalysts for the Henry reaction. Our
EMDee is well-known as one way to determine the % ee of a
chiral compound,57-60 based on the enantioselectivity of enzy-
matic kinetic resolution and a probe molecule to evaluate the
activity. Because the reactivity of the enzyme is affected by the
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Figure 4. HTS of candidate kinetic resolution catalysts: 1 mM 6 was acylated with 5 mM acid anhydride in the presence of 5 mM catalyst in 300 μL of
various solvents. Reaction mixtures were diluted 100 times with DMSO for measurement of fluorescence.
reaction mixture would reflect the % ee. Compound 6 with
various values of % ee was prepared by mixing solutions of (R)-6
and (S)-6, followed by treatment with (R)- or (S)-12 and
isobutyric anhydride in toluene, and the fluorescence intensity
of the reaction mixture was measured. The relation between
fluorescence intensity and % ee is illustrated in Figure 6 for (R)-
12 and Figure 7 for (S)-12. (R)-12 with isobutyric anhydride in
toluene readily acylates (R)-6 to produce nonfluorescent 8, while
it hardly acylates (S)-6, so the fluorescence intensity of the
reaction mixture indicates the concentration of (S)-6 which has
not reacted. On the other hand, when (S)-12 is used as the
catalyst, the fluorescence intensity of the reaction mixture
indicates the concentration of (R)-6 which has not reacted.
Namely, the fluorescence decrease at the second step for (R)-
and (S)-12 was measured and the sum of the fluorescence
decreases in both reaction mixtures reflects the reaction yield
of the first step, whereas the difference of fluorescence decrease
between the two reaction mixtures reflects the % ee.
Table 1. Enantioselectivity of (R)-12 with Various Acid
Anhydrides in Toluenea
entry
1
acid anhydride
yieldb (%) eeb (%) selectivity average
44.2
46.0
46.3
35.4
35.5
34.2
31.7
31.7
31.8
63.1
72.1
72.1
49.8
49.9
48.5
44.1
45.0
44.7
16.8
25.9
24.3
34.6
33.8
47.0
59.2
105.6
76.2
propionic anhydride
22.3
38.5
80.3
2
butyric anhydride
3
isobutyric anhydride
a 1 mM racemic 6, 0.5 mM acid anhydride and 0.5 mM (R)-12 were
reacted in 1 mL of toluene at room temperature for 90 min. b Yield and
ee were determined by HPLC from the peak area with absorbance
detection at 500 nm.
In both Figures 6 and 7, the fluorescence measurement error at
higher fluorescence intensities is greater than that at lower
fluorescence intensities. The reason for this is that at lower
fluorescence intensities, the reaction is almost complete and the
product 8 is predominant, so that the fluorescence intensity is
stable. On the other hand, at higher fluorescence intensities, the
substrate 6 is still predominant, the reaction is progressing, and
so the fluorescence intensity is changing.
In this system, the solvent, catalyst, and nitromethane used in
the first step would be present as impurities in the second step.
Therefore, we confirmed that % ee determination at the second
step is not affected by such impurities. As a model first-step
reaction, we used 5 mM 5, 100 equiv of nitromethane, 0.1 equiv
of TEA as a catalyst, and DMF as a solvent, and the reaction
chirality of the substrate, % ee of the substrate can be estimated
from enzyme activity measured with the probe molecule. How-
ever, EMDee can estimate the amount of only one enantiomer;
this is an essential limitation of enzymatic kinetic resolution, so a
nonenzymatic kinetic resolution is desirable.61
Since we found that (R)-12 with isobutyric anhydride in
toluene is a superior kinetic resolution condition for nitroaldol
among the conditions tested in Figure 4, it should be possible to
establish an HTS system for % ee determination with (R)-12 and
(S)-12. (R)-12 is commercially available, but (S)-12 is not, so we
synthesized (S)-12 according to a conventional method em-
ployed to obtain (R)-12 (see the Supporting Information).62 We
examined whether or not the fluorescence intensity of the
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Figure 5. In situ reaction monitoring: 10 μM of (R)- or (S)-6 was acylated with 20 mM acid anhydride in the presence of 20 mM (R)-12 in 3 mL of
toluene. The blue line shows the relative fluorescence intensity of (R)-6, the red line shows the relative fluorescence intensity of (S)-6, and the black line
shows the relative fluorescence intensity of control (no catalyst). Propionic anhydride (b), butyric anhydride (2), and isobutyric anhydride (9) were
used. The reaction was performed in a fluorescence spectrometer, and fluorescence intensity was measured every 0.5 s (Ex 500 nm, Em 515 nm).
Scheme 4. HTS Procedure for Asymmetric Catalyst of Henry Reaction
mixture of the first step was diluted 10-fold with toluene solution
containing (R)-12 and isobutyric anhydride. Thus, compound 6
with various % ee values was treated with (R)-12 and isobutyric
anhydride in toluene containing DMF, nitromethane, and TEA
as impurities. After the reaction, the fluorescence intensity of the
reaction mixture was measured (Figure 8). Similarly to the case
of Figure 6, the fluorescence intensity was well correlated with
% ee. These results confirmed that our HTS system can evaluate
yield and % ee based on measurement of fluorescence.
resolution may also be useful for discovery of superior asym-
metric catalysts for other reactions.
’ EXPERIMENTAL SECTION
UV/vis and fluorescence analysis. For absorption or fluores-
cence measurement, compounds were dissolved in DMSO to obtain
1 mM stock solutions. These stock solutions were diluted with organic
solvents as specified in the figure legends to the desired concentration.
For determination of the fluorescence quantum yield (Φfl), fluorescein
in 100 mM aq NaOH (Φfl = 0.85) was used as a fluorescence standard.
Fluorescence-Based HTS Assay for Determination of the
Reaction Yield. To determine the reaction yield, a calibration curve of
reaction yield versus fluorescence intensity was prepared. For preparation
of the calibration curve, solutions of 6and 8 (10 μM total concentration) in
DMSO were mixed to obtain standard solutions corresponding to 0%,
20%, 40%, 60%, 80%, and 100% yields, respectively; these solutions were
excited at 500 nm, and the fluorescence intensity was measured at 515 nm.
For the HTS assay, 1 mM 6 was acylated with acid anhydride (5 equiv) in
the presence of catalyst (5 equiv) in 300 μL of various solvents at room
temperature for 1 h. Reaction mixtures were diluted 100 times with DMSO
for measurement of fluorescence. The reaction yields were obtained from
the calibration curve by interpolation of the measured fluorescence
intensity of the samples.
’ CONCLUSION
We have developed an HTS system for kinetic resolution
catalysts of nitroaldol, employing a rationally designed fluores-
cence probe whose fluorescence is controlled by PET and
changes upon reaction. With this HTS system, both reactivity
and enantioselectivity could be evaluated simply by measuring
the fluorescence intensity. Our novel HTS system revealed that
(R)-12 with isobutyric anhydride in toluene is a superior kinetic
resolution condition. (R)-12 with isobutyric anhydride in to-
luene acylated the hydroxyl group of (R)-6 80 times faster than
that of (S)-6. Furthermore, we established an HTS system for
asymmetric catalysts of the Henry reaction, employing (R)- or
(S)-12. Such HTS systems based on nonenzymatic kinetic
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Figure 6. HTS system for % ee determination: 1 mM 6 (note that here 100% ee corresponds to (S)-6, while -100% ee corresponds to (R)-6) was
treated with 3 mM (R)-12 and 3 mM isobutyric anhydride in 1 mL of toluene at room temperature for 1 h. The reaction mixture was diluted 100 times
with DMSO, and the fluorescence intensity was measured (Ex 500 nm, Em 515 nm) (n = 3).
Synthesis of 4-Hydroxymethylbenzaldehyde (1). Ter-
ephthalaldehyde (6.7 g, 50 mmol) was dissolved in 100 mL of dry
THF. NaBH4 (437 mg, 12.5 mmol) was added, and the solution was
stirred at room temperature with a CaCl2 tube for 1 h. The solvent was
evaporated under reduced pressure. The residue was diluted with
CH2Cl2 and washed with water 3 times. The organic solution was
washed with brine and dried over Na2SO4. The compound was purified
by silica gel column chromatography (eluent: CH2Cl2/MeOH = 19/1)
to give a colorless oil (1.7 g, 25%). 1H NMR (300 MHz, CDCl3): δ 2.09
(s, 1H), 4.74 (s, 2H), 7.54 (d, 2H, J = 8.1 Hz), 7.88 (d, 2H, J = 8.1 Hz),
10.01 (s, 1H).
Synthesis of 4-Acetoxymethylbenzaldehyde (2). Com-
pound 1 (1.7 g, 12.5 mmol) was dissolved in 50 mL of dry pyridine, and
3 mL of acetic anhydride was added at 0 °C for 12 h. The reaction
mixture was diluted with CH2Cl2, washed with water three times and
brine once, dried over Na2SO4, filtered, and evaporated. The residue was
purified by silica gel column chromatography (eluent: CH2Cl2) to give a
colorless oil (1.5 g, 67%). 1H NMR (300 MHz, CDCl3): δ 2.15 (s, 3H),
5.19 (s, 2H), 7.52 (d, 2H, J = 8.0 Hz), 7.89 (d, 2H, J = 8.0 Hz), 10.03 (s,
1H). 13C NMR (75 MHz, CDCl3): δ 20.9, 65.4, 128.2, 130.0, 136.1,
142.7, 191.7.
atmosphere. One drop of TFA was added, and the solution was stirred
at room temperature for 12 h. A solution of DDQ (545 mg, 2.0 mmol) in
CH2Cl2 was added, and stirring was continued for 20 min. The reaction
mixture was washed with water three times and brine once, dried over
Na2SO4, filtered, and evaporated. The compound was purified by short
column chromatography over alumina (eluent: CH2Cl2). The brown
powder thus obtained and 5 mL of DIEA were dissolved in 200 mL of
toluene under an Ar atmosphere. Then 5 mL of BF3 Et2O was added,
3
and the solution was stirred at room temperature for 30 min. The
reaction mixture was washed with water three times and brine once,
dried over Na2SO4, filtered, and evaporated. The residue was purified by
silica gel column chromatography (eluent: CH2Cl2/n-hexane = 1/1) to
give an orange solid (202 mg, 26%). 1H NMR (300 MHz, CDCl3): δ
1.37 (s, 6H), 2.16 (s, 3H), 2.55 (s, 6H), 5.20 (s, 2H), 5.98 (s, 2H), 7.28
(d, 2H, J = 8.2 Hz), 7.47 (d, 2H, J = 8.2 Hz). 13C NMR (75 MHz,
CDCl3): δ 14.4, 14.6, 21.0, 65.6, 121.2, 128.2, 128.4, 131.4, 134.8, 137.1,
141.2, 143.0, 155.6, 170.7. HRMS (ESIþ): calcd for ([M þ Na]þ)
419.1718, found 419.1730. Anal. Calcd for C22H23BF2N2O2: C, 66.69;
H, 5.85; N, 7.07. Found: C, 66.63; H, 5.80; N, 7.20.
Synthesis of 8-(4-Hydroxymethylphenyl)-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (4). Com-
pound 3 (200 mg, 0.51 mmol) was dissolved in 5 mL of CH2Cl2, and
then 10 mL of MeOH and 2 mL of NaOH aq (0.1 M) were added. The
reaction mixture was stirred at room temperature for 1 h, diluted with
CH2Cl2, washed with water three times and brine once, dried over
Synthesis of 8-(4-Acetoxymethylphenyl)-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (3). Com-
pound 2 (356 mg, 2.0 mmol) and 2,4-dimethylpyrrole (380 mg, 4.0
mmol) were dissolved in 350 mL of dry CH2Cl2 under an Ar
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Figure 7. HTS system for % ee determination: 1 mM 6 (note that here 100% ee corresponds to (S)-6, while -100% ee corresponds to (R)-6) was
treated with 3 mM (S)-12 and 3 mM isobutyric anhydride in 1 mL of toluene at room temperature for 1 h. The reaction mixture was diluted 100 times
with DMSO, and the fluorescence intensity was measured (Ex 500 nm, Em 515 nm) (n = 3).
Na2SO4, filtered, and evaporated. The residue was purified by silica gel
column chromatography (eluent: CH2Cl2) to give an orange solid (217
mg, quant). 1H NMR (300 MHz, CDCl3): δ 1.38 (s, 6H), 1.93 (t, 1H,
J = 5.7 Hz), 2.55 (s, 6H), 4.79 (d, 2H, J = 5.7 Hz), 5.98 (s, 2H), 7.27 (d,
2H, J = 8.4 Hz), 7.49 (d, 2H, J = 8.4 Hz). 13C NMR (75 MHz, CDCl3): δ
14.4, 14.5, 64.7, 121.2, 127.3, 128.1, 131.4, 134.2, 141.5, 141.9, 143.1,
155.4. HRMS (ESIþ): calcd for ([M þ Na]þ) 377.1613, found
377.1660. Anal. Calcd for C20H21BF2N2O: C, 67.82; H, 5.98; N, 7.91.
Found: C, 68.00; H, 6.07; N, 7.94.
Synthesis of 8-(4-Formylphenyl)-4,4-difluoro-1,3,5,7-tet-
ramethyl-4-bora-3a,4a-diaza-s-indacene (5). Compound 4
(100 mg, 0.28 mmol) was dissolved in 50 mL of CH2Cl2 and PCC
(120 mg, 0.56 mmol) was added, followed by 120 mg of MgSO4. The
reaction mixture was stirred at room temperature under an Ar atmo-
sphere for 4 h, filtered through Celite, and purified by silica gel column
chromatography (eluent: CH2Cl2) to give an orange solid (68 mg, y.
68%). 1H NMR (300 MHz, CDCl3): δ 1.35 (s, 6H), 2.56 (s, 6H), 6.00
(s, 2H), 7.50 (d, 2H, J = 8.1 Hz), 8.03 (d, 2H, J = 8.1 Hz), 10.11 (s, 1H).
13C NMR (75 MHz, CDCl3): δ 14.4, 14.5, 121.6, 129.1, 130.3, 130.7,
136.6, 139.6, 141.3, 142.7, 156.1, 191.4. HRMS (ESIþ): calcd for ([M þ
Na]þ) 375.1456, found 375.1470. Anal. Calcd for C20H19BF2N2O: C,
68.21; H, 5.44; N, 7.95. Found: C, 68.23; H, 5.53; N, 7.92.
dissolved in 20 mL of CH2Cl2, and then 20 mL of MeOH and 0.1 mL of
NaOH aq (2 M) were added at 0 °C. The reaction mixture was stirred at
0 °C for 4 h, diluted with CH2Cl2, washed with water three times and brine
once, driedoverNa2SO4, filtered, and evaporated. The residue was purified
by silica gel column chromatography (eluent: CH2Cl2) to give an orange
solid (1.2 g, yield 93%). 1H NMR (300 MHz, CDCl3): δ 1.34 (s, 6H), 2.55
(s, 6H), 3.01 (d, 1H, J = 4.2 Hz), 4.56 - 4.70 (m, 2H), 5.53 - 5.58 (m,
1H), 5.99 (s, 2H), 7.35 (d, 2H, J = 7.9 Hz), 7.55 (d, 2H, J = 7.9 Hz). 13C
NMR (75 MHz, CDCl3): δ 14.4, 14.6, 70.5, 81.2, 121.4, 126.6, 128.8,
131.3, 135.8, 139.1, 140.5, 142.8, 155.8. HRMS (ESIþ): calcd for ([M þ
Na]þ) 436.1620, found 436.1651. Anal. Calcd for C21H22BF2N3O3: C,
61.04; H, 5.37; N, 10.17. Found: C, 60.98; H, 5.37; N, 10.08.
Synthesis of 8-(4-((E)-2-Nitrovinyl)phenyl)-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (8). Com-
pound 6 (41 mg, 0.1 mmol) was dissolved in 10 mL of dry CH2Cl2, and
then 2 mL of Ac2O and 2 mL of dry pyridine were added. The reaction
mixture was stirred at room temperature for 4 h, washed with water three
times and brine once, dried over Na2SO4, filtered, and evaporated. The
residue was purified by silica gel column chromatography (eluent:
CH2Cl2) to give a red solid (33 mg, yield 86%). 1H NMR (300 MHz,
CDCl3): δ 1.39 (s, 6H), 2.57 (s, 6H), 6.00 (s, 2H), 7.42 (d, 2H, J = 8.1
Hz), 7.66 (d, 1H, J = 13.7 Hz), 7.70 (d, 2H, J = 8.1 Hz), 8.06 (d, 1H, J =
13.7 Hz). 13C NMR (75 MHz, CDCl3): δ 14.6, 14.7, 121.6, 129.5, 129.7,
130.8, 131.0, 137.9, 138.0, 139.1, 139.7, 142.7, 156.2. HRMS (ESIþ):
calcd for ([M þ Na]þ) 418.1514, found 418.1514. Anal. Calcd for
Synthesis of 8-(4-(1-Hydroxy-2-nitroethyl)phenyl)-4,4-di-
fluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene
(6). Compound 5 (1.1 g, 3.1 mmol) and 3 mL of nitromethane were
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The Journal of Organic Chemistry
FEATURED ARTICLE
Figure 8. Effect of impurities on evaluation of % ee. A solution of 0.5 mM 6 (note that here 100%ee corresponds to (S)-6, while -100% ee corresponds
to (R)-6) was treated with 5 mM (R)-12 and 10 mM isobutyric anhydride in 1 mL of toluene solution which contained 10% DMF, 50 mM nitromethane,
and 0.05 mM TEA as impurities at room temperature for 1 h. The reaction mixture was diluted 50 times with DMSO, and the fluorescence intensity was
measured (Ex 500 nm, Em 15 nm) (n = 3).
C21H20BF2N3O2: C, 63.82; H, 5.10; N, 10.63. Found: C, 63.53; H,
5.30; N, 10.54.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: tlong@mol.f.u-tokyo.ac.jp.
Synthesis of (R)-8-(4-(1-Hydroxy-2-nitroethyl)phenyl)-
4,4-difluoro-2-iodo-1,3,5,7-tetramethyl-4-bora-3a,4a-dia-
za-s-indacene ((R)-9). (R)-6 (51.3 mg, 0.12 mmol) and I2 (15.7 mg,
0.062 mmol) were dissolved in 10 mL of CH2Cl2, and then 20 mL of
EtOH was added followed by HIO3 (10.9 mg, 0.062 mmol) dissolved in
10 mL of H2O. The reaction mixture was stirred at 50 °C for 20 min and
cooled to room temperature, and then 20 mL of saturated Na2S2O3 aq
was added and the whole was extracted with CH2Cl2. The organic layer
was washed with water three times and brine once, dried over Na2SO4,
filtered, and evaporated. The residue was purified by silica gel column
chromatography (eluent: CH2Cl2) to give a red solid (56.4 mg, yield
84%). 1H NMR (300 MHz, CDCl3): δ 1.35 (s, 6H), 2.57 (s, 3H), 2.63
(s, 3H), 3.02 (d, 1H, J = 4.4 Hz), 4.60 (dd, 1H, J = 13.2 Hz, 3.7 Hz), 4.67
(dd, 1H, J = 13.2 Hz, 8.0 Hz), 5.55 - 5.60 (m, 1H), 6.06 (s, 1H), 7.34 (d,
2H, J = 8.1 Hz), 7.57 (d, 2H, J = 8.1 Hz). 13C NMR (75 MHz, CDCl3): δ
14.7, 14.8, 15.8, 16.7, 70.5, 81.2, 84.5, 122.5, 126.8, 128.7, 130.8, 131.7,
135.6, 139.4, 140.3, 143.0, 144.8, 154.9, 158.2. HRMS (ESI-): calcd for
([M - H]-) 538.0611, found 538.0606. Anal. Calcd for C21H21BF2I-
N3O3: C, 46.78; H, 3.93; N, 7.79. Found: C, 46.43; H, 3.96; N, 7.67.
’ ACKNOWLEDGMENT
We thank Nobuko Kanehisa, Mihoko Maruyama, and Shigeru
Sugiyama, Osaka University and CREST JST; Hiroaki Adachi,
Kazufumi Takano, Hiroyoshi Matsumura, and Tsuyoshi Inoue,
Osaka University, SOSHO Inc., and CREST JST; and Satoshi
Murakami, Tokyo Institute of Technology, SOSHO Inc., and
CREST JST, for experimental support. This research was supported
in part by the Science and Technology Incubation Program of the
Japan Science and Technology Agency. This work was also
supported in part by a Grant-in-Aid for JSPS Fellows (to T.M.),
by the Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan (Grant Nos. 19205021 and 20117003 to Y.U.), and
by Industrial Technology Research Grant Program in 2009 from
New Energy and Industrial Technology Development Organization
(NEDO) of Japan (to T.T.). We also thank Mr. Takashi Tsujino,
Science Graphics for his assistance in creating the cover art.
’ ASSOCIATED CONTENT
’ REFERENCES
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