2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of β-d-galactosidase activities

Article abstract of DOI:10.1016/j.bmcl.2013.01.043

2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives were synthesized as novel artificial fluorescent pigment dyeing substrates for β-d-galactosidase. The substrates, which exhibited non-fluorescence or weak fluorescence in solution phase, were

Full text of DOI:10.1016/j.bmcl.2013.01.043

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2245–2249
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-(Benzothiazol-2-yl)-phenyl-b-D-galactopyranoside derivatives
as fluorescent pigment dyeing substrates and their application
for the assay of b-D-galactosidase activities
b
a
b
b
Tadamune Otsubo a, , Akira Minami , Haruna Fujii , Risa Taguchi , Tadanobu Takahashi ,
⇑
Takashi Suzuki b, Fumiteru Teraoka a, Kiyoshi Ikeda a
a Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1 Hirokoshingai, Kure, Hiroshima 737-0112, Japan
b Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
2-(Benzothiazol-2-yl)-phenyl-b-
rescent pigment dyeing substrates for b-
D
-galactopyranoside derivatives were synthesized as novel artificial fluo-
-galactosidase. The substrates, which exhibited non-fluores-
cence or weak fluorescence in solution phase, were smoothly hydrolyzed by b- -galactosidase from
Received 2 December 2012
Revised 10 January 2013
Accepted 12 January 2013
Available online 4 February 2013
D
D
Aspergillus oryzae and yielded a water-insoluble strong fluorescent pigment. The difference of fluorescent
intensity exhibited a linear relationship with the amount of enzyme.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
2-(Benzothiazol-2-yl)-phenyl-b-
galactopyranoside derivative
Fluorescent pigment dye
D-
b-D-galactosidase
Aspergillus oryzae
Fluorescent stain
One of the most important reporter proteins, widely used in
gene expression and regulation, is b- -galactosidase. It is used
fluorescent hydrolysis product tends to diffuse through the cell
membrane or to the culture solution.13 The cause of this problem
is that the observation of fluorescence is generally possible only
in a solution state, so another way to resolve this issue is fluores-
cent pigment dyeing substrate. The fluorescent pigment dyeing
substrates will be soluble to water, and yield a water-insoluble
fluorescent pigment upon enzymatic hydrolysis. The yielded
fluorescent particles are immobile at the reaction point and stain
tissue without diffusing to the culture solution. Against this back-
ground, we have been focusing on 2-(benzothiazol-2-yl)-phenol
derivatives 1.14
We have been interested in their strong fluorescent property,
associated with a large Stokes shift (150 nm ꢀ) based on the
excited-state intramolecular proton transfer (ESIPT) mechanism.15
This solid fluorescent property makes it easy to detect a very small
amount of fluorophor or low enzyme activity because of the local
high dye concentration in the solid state resulting in a bright lumi-
nescent particle. Solid state analysis will make it possible to avoid
the influence of solvent polarity or pH, and to stain living cells with
a fluorescent pigment without fixing. Given the large Stokes shift,
the self-absorption of fluorescence could be avoided, and it also be-
comes unnecessary to separate exciting light and emitting light.
This enables the choice of a convenient wavelength and high-
precision measurement.
D
with many substrates designed for various detection methods,
such as colorimetric,1 chromogenic,2 radioactive,3 nuclear mag-
netic resonance,4 and fluorescent methods.5 The chromogenic
method is commonly used for histochemical staining. For exam-
ple, X-Neu5Ac, from which X–OH (5-bromo-4-chloro-3-hydrox-
yindole) would be yielded upon enzymatic hydrolysis and air-
oxidized to water-insoluble indigo blue, has been used for imag-
ing of sialidase activities from influenza virus.6 The fluorescence
imaging methods are superior in terms of polarity or metabolism
selectivity, ease of use, time required, and cost. In addition, fluo-
rescent probes are sensitive enough to detect even a single mol-
ecule,7 and enable high-resolution microanalysis8 or bio-
imaging;9 however, most are also associated with a variety of dis-
advantages. For example, fluorescein di-b-D-galactopyranoside
(FDG)10 must be loaded into cells by hypotonic shock11 or applied
to fixed or permeabilized cells12 because of its poor cellular per-
meability. On the other hand, high cellular permeability results in
indistinct fluorescent imaging because an enzymatically yielded
⇑
Corresponding author. Tel.: +81 823 73 8932; fax: +81 823 73 8981.
E-mail address: t-ootubo@ps.hirokoku-u.ac.jp (T. Otsubo).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.043

2246
T. Otsubo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2245–2249
spectra in water are indicated in Fig. 2B, which were measured
R3
1a: R1 = H
, R2 = H , R3 = H
OH
R2
S
with the existence of particulates because of their poor solubility
to water. Their absorption-maximum wavelengths were in the nar-
row range from 353 to 372 nm. That of 1b was 353 nm and those of
the others were 372 nm. On the other hand, fluorescent-maximum
wavelengths showed large Stokes shifts, which were in the range
from 150 to 200 nm, and their fluorescent wavelengths were over-
lapped with the main visible regions.
1b: R1 = OMe, R2 = H , R3 = H
N
R
1 = H
, R2 = Br , R3 = H
, R2 = OMe, R3 = H
, R2 = OMe, R3 = Cl
1c:
1d: R1 = H
1e: R1 = H
R1
Figure 1. Selected 2-(benzothiazol-2-yl)-phenol derivatives 1.
The fluorescent pigment substrates 2a–2e were prepared as
In this letter, we report the synthesis of novel fluorescent pig-
ment substrates for b- -galactosidases and their response to b-
galactosidase from Aspergillus oryzae.
shown in Scheme 1. According to
a procedure previously
reported,17 the core fluorescent pigment dyes 1a–1e were success-
fully synthesized from corresponding salicylaldehydes (3a–3d) and
2-aminothiophenols (4a–4b) in yields ranging from 52% to 71%.
D
D-
At first, we selected 1a–1e16 (Fig. 1) as the fluorescent pigment
dyes for the synthetic substrates 2a–2e. As indicated in Figure 2, by
adjusting the electronic character of phenol or benzothiazole ring,
their fluorescent wavelength in the solid state could be controlled.
Relative to 1a with no substituent, an electron-donating effect at
the meta-position of phenolic hydroxyl moiety caused a shift of
the fluorescent wavelength to a shorter one (Fig. 2, green 1a vs blu-
ish green 1b). On the other hand, the electron-donating effect by
the lone pair from the para-position made the fluorescent wave-
length shift toward the longer side (Fig. 2, green 1a vs yellow green
1c, orange 1d). In addition, the reduction of electron density of the
benzothiazol ring by the introduction of a chlorine atom at 5-posi-
tion caused a further shift to the longer-wavelength end (Fig. 2, or-
ange 1d vs reddish 1e). Their UV–VIS absorption and fluorescence
Dyes 1a–1e were coupled with 2,3,4,6-tetra-O-acetyl-
pyranosyl bromide (5) and converted to 2-(benzothiazol-2-yl)-
phenyl-2,3,4,6-tetra-O-acetyl-b- -galactopyranoside derivatives
D-galacto-
D
6a–6e under sodium hydride in the THF–DMF condition in the
range of yields of 16–89%.
The pro-substrates 6a–6e exhibited no or slight fluorescence in
solid condition and solution (Fig. 3A,B). In solid state, 6 exhibited
slight fluorescence, but in solution 6d and 6e exhibited weak pur-
ple fluorescence, which was quite different from the original color
of its aglycone. Notably, 6a and 6c exhibited no fluorescence at all.
All acetyl groups of 6 were easily removed using sodium methox-
ide in methanol, and the fluorescent pigment substrates 2a–2e
were given in yields ranging from 79% to 96%. The substrates
A
B
Absorbance
372
1a: 518
1d: 585
1a
1b
1c
1d
1e
1c: 526
1b: 503
353
1e: 594
370
250
300
350
400
450
500
550
600
650
700
wavelength (nm)
Figure 2. Properties of fluorescent pigment dyes 1a–1e. (A) Various color luminescence under excitation at 352 nm in a solid state (1.1 0.1 mg). (B) Absorbance (dashed
lines) and fluorescent (solid lines) spectrums of fluorescent pigment dyes 1a–1e in aqueous phosphorous buffer (pH 7.4). Numbers represent wavelength (nm) at peak
absorbance or fluorescence intensity. Ri: relative absorbance or fluorescence intensity.

T. Otsubo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2245–2249
2247
R2
R1
O
S
O
N
RO
RO
R3
OR
OR
SH
R3
OH
R2
6a: R1 = H
, R2 = H , R3 = H , R = Ac
NH2
CHO
6b: R1 = OMe, R2 = H , R3 = H , R = Ac
b
a
6c: R1 = H
6d: R1 = H
6e: R1 = H
, R2 = Br , R3 = H , R = Ac
, R2 = OMe, R3 = H , R = Ac
, R2 = OMe, R3 = Cl, R = Ac
1a~1e
R1
O
Br
AcO
AcO
c
3a: R1 = H
, R2 = H
4a: R3 = H
OAc
3b: R1 = OMe, R2 = H
4b: R3 = Cl
OAc
2a: R1 = H
, R2 = H , R3 = H , R = H
3c: R1 = H
3d: R1 = H
, R2 = Br
2b: R1 = OMe, R2 = H , R3 = H , R = H
, R2 = OMe
2c: R1 = H
2d: R1 = H
2e: R1 = H
, R2 = Br , R3 = H , R = H
, R2 = OMe, R3 = H , R = H
, R2 = OMe, R3 = Cl, R = H
Scheme 1. Synthetic route for fluorescent pigment probes 2a–2e. Conditions: (a) EtOH–THF, 80 °C, 18 h, yield: 67% for 1a, 52% for 1b, 57% for 1c, 61% for 1d, and 71% for 1e.
(b) 5, NaH, THF–DMF, 75 °C, 18 h, yield: 40% for 6a, 16% for 6b, 62% for 6c, 89% for 6d, and 68% for 6e. (c) NaOMe, MeOH–THF, room temperature, 1 h, yield: 79% for 2a, 96% for
2b, 92% for 2c, 91% for 2d, and 61% for 2e.
Figure 3. The fluorescent properties of 6a–6e and 2a–2e. Conditions: All pictures were taken under excitation condition at 352 nm. (A, C): 1.1 0.1 (mg) Solvent free
condition (B, D) 1.1 0.1/550 (mg/lL) in CDCl3 and DMSO-d6 solution, respectively.
exhibited fluorescence very weakly or not at all in a solid state
(Fig. 3C) and, compared with dyes 1a–1e, the differences were
clear. However, in solution (Fig. 3D), 2a was non-fluorescent, and
the others exhibited only weak purple fluorescence. This finding
suggested that the interruption of the ESIPT mechanism had
caused a small Stokes shift.
Michaelis constant (Km) and maximum velocity (Vmax) were
7.2 Â 10À4
M and 86.7 lmol/min/mg with o-nitrophenyl-b-D-
galactopyranoside and 1.8 Â 10À2 M and 121.9
lmol/min/mg with
A citric acid-phosphoric acid buffer
lactose, respectively.18
(100 mM, pH 4.5) solution containing 10 unit/mL enzyme and
100 mol/L 2 was prepared, and incubated at 28 °C for 1 h. With-
l
Qualitative analysis was carried out in order to confirm the re-
sponse of fluorescent pigment substrates 2a–2e to a b-D-galacto-
sidase from Aspergillus oryzae (Fig. 4A). It was reported that the
enzyme was stable in the pH range from 4.0 to 9.0, and its
out quenching the enzyme reaction, the hydrolysis of substrates 2
was confirmed by the observation of intense fluorescent pigment
with a large Stokes shift under excitation at 352 nm. This fluores-
cent color change with a large Stokes shift made it unnecessary to

2248
T. Otsubo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2245–2249
A
B
Figure 4. Results of enzymatic reactions of 2a–2e with b-
response of substrate to enzyme by the naked eye under black light (352 nm) irradiating conditions after incubation in 100 mM citric acid–phosphoric acid buffer (pH 4.5) at
28 °C for 1 h [substrate ( mol/L), enzyme (unit/mL)]: (a) = [100, 0] (b) = [100, 10]. (B): The differences (circles) of fluorescent intensity ( FI) from the background were
calculated from the average of three measurements, and solid lines are the results of linear regression analysis for each substrate. The fluorescent measurements took place
after quenching the enzymatic reactions by the addition of aqueous 1 M Na2CO3 solution to the enzyme reaction mixture, which had been incubated with 10 mol/L substrate
D-galactosidase from Aspergillus oryzae. (A) Each picture is the result of a qualitative experiment to determine the
l
D
l
and a corresponding amount of enzyme in 100 mM citric acid–phosphoric acid buffer (pH 4.5) at 37 °C for 1 h. Intensity of fluorescence was measured with a 96-well
microplate reader (ex/em: 370 nm/520 nm for 2a, 354 nm/505 nm for 2b, 372 nm/526 nm for 2c, 374 nm/582 nm for 2d, and 374 nm/596 nm for 2e).
compare the fluorescent intensity before and after the enzyme
reaction. And the clear color change was effective to avoid an
influence of remaining substrate or the need to increase the mea-
surement accuracy. Even under low pH conditions (pH 4.5), at
recognize 10 unit/mL enzyme activity. Generally speaking, fluo-
rescent analysis is greatly influenced by the polarity or pH. For
example, 4-methylumbelliferone (pKa = 7.79)19 would exhibit
only weak fluorescence at pH 4.5. It is a great advantage of a fluo-
rescent pigment dye that the fluorescent observation was possi-
ble under low pH conditions.
100
lmol/L substrate, intense fluorescence was observed. In addi-
tion, the on/off ratio of fluorescent intensity was high enough to

T. Otsubo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2245–2249
2249
5. (a) Jurkevica, A.; Halsall, H. B.; Seliskar, C. J.; Heineman, W. R. Anal. Bioanal.
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Linear regression analysis was also carried out for each sub-
strate with 100 L of reaction buffer (100 mM citric acid—phos-
phoric acid, pH 4.5) containing 10 mol/L fluorescent pigment
l
l
substrate 2 on a 96-well microplate reader. Although the fluores-
cence intensity was observed from a precipitated pigment upon
hydrolysis, the difference of fluorescent intensity indicated a linear
relationship against the amount of enzyme (Fig. 4B). The fluores-
cent color change with a large Stokes shift would be effective to
avoid an influence of remaining substrate or increase measure-
ment accuracy. This linearity also gave a guarantee of the concise
quantitative analysis of enzyme activity. The trends were large en-
ough for application to the measurement of enzyme activity. The
trend for the regression line of 2a was about 3–5 times steeper
than those of the others. These results suggested that 2a would
be suitable for highly precise enzyme analysis and imaging. The
others would be superior in terms of fluorescent multicolor stain-
ing and high stainability. The increase of stainability brought about
by the introduction of a halogen atom on the aromatic ring, result-
ing in lower solubility to water than that of 2a.
We synthesized novel fluorescent pigment substrates for b-D-
galactosidase,20–22 and 2a exhibited no fluorescence in solution,
while the others exhibited only weak purple fluorescence. The
fluorescent pigment substrates 2 were smoothly hydrolyzed by
14. (a) Santra, M.; Roy, B.; Ahn, K. H. Org. Lett. 2011, 13, 3422; (b) Chen, W.; Xing,
Y.; Pang, Y. Org. Lett. 2011, 13, 1362; (c) Kim, T.; Kang, H. J.; Han, G.; Chung, S. J.;
Kim, Y. Chem. Commun. 2009, 5895; (d) Sun, W.; Li, W.; Li, J.; Zhang, J.; Du, L.; Li,
M. Tetrahedron Lett. 2012, 53, 2332.
b-D
-galactosidase from Aspergillus oryzae.23 With the formation of
a fluorescent pigment, various intense colors of fluorescence were
recovered. Their ratios of fluorescence intensity before and after
enzyme reaction were high enough to recognize enzyme activity
by the naked eye without a quenching enzyme reaction. In addi-
tion, it should be mentioned that the enzymatic hydrolysis was
confirmed at pH 4.5. The difference of fluorescent intensity showed
a linear relationship against the enzyme concentration range of our
experiments. Because of immobility or poor solubility to water,
fluorescent pigment staining with 1 is superior in terms of not only
the possibility of observing fluorescence at low pH, but also in that
it is possible to stain live cells on the outer surface or interior of a
cell membrane without fixing or permeabilizing.
The following further examinations are now in progress; the
investigation of selective response of 2a–2e to b-D-galactosidase
from Escherichia coli or other eukaryotic cells, the staining of live
cells, and the synthesis of fluorescent pigment substrates for other
enzymes.
15. (a) Cellier, M.; Fabrega, O. J.; Fazacherley, E.; James, A. L.; Orenga, S.; Perry, J. D.;
Salwatura, V. L.; Stanforth, S. P. Bioorg. Med. Chem. Lett. 2011, 19, 2903; (b) dos
Santos, R. C.; Faleiro, N. V. S.; Campo, L. F.; Scroferneker, M. L.; Carbellini, V. A.;
Rodembusch, F. S.; Sterani, V. Tetrahedron Lett. 2011, 52, 3048; (c) Chuang, W.;
Hsieh, C.; Lai, C.; Lai, C.; Shih, C.; Cehn, K.; Hung, W.; Shu, Y.; Chou, P. J. Org.
Chem. 2011, 76, 8189.
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D. C.; Friedman, B. L.; Pastoriza-Munoz, E. P. Anal. Biochem. 1986, 156, 202.
20. General procedure for synthesis of 2-benzothiazol-2-yl phenol derivatives (1).
Synthesis of 1e is typical.
A solution of 5-methoxysalicylaldehyde (1.52 g,
10 mmol) and 2-amino-4-chlorothiophenol in THF–EtOH (10 mL/10 mL) was
stirred at 80 °C for 24 h. After cooling to room temperature, the solvent was
removed under reduced pressure, and the resulting precipitation was
suspended in Et2O, filtered, washed with Et2O, and dried to give desired
product 1e (2.06 g, 71% yield) as orange needles.
21. General procedure for synthesis of 2-(benzothiazol-2-yl)-phenyl-2,3,4,6-tetra-O-
acetyl-b-D-galactopyranoside (6): Synthesis of 6a is typical. To a suspension of
NaH (60% in mineral oil, 60 mg, 1.5 mmol) in THF (7 mL) was added dropwise a
solution of 5 (412 mg, 1 mmol) and 1a (227 mg, 1 mmol) in THF (3 mL) under
an argon atmosphere, and the resulting reaction mixture was stirred at 75 °C
for 18 h. After cooling to room temperature, the reaction mixture was diluted
with AcOEt, and the organic layer was washed successively with water (3
times) and brine, dried over dry Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (n-hexane/AcOEt = 2:1 to 1:1 as an eluent) to give the
desired product 6a (220 mg, 40% yield) as a colorless amorphous solid after
freeze–drying from dioxane.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.043. These data include MOL files and InChiKeys of the most
important compounds described in this article.
22. General procedure for synthesis of (2-benzothiazol-2-yl)-phenyl-b-D-
References and notes
galactopyranoside derivatives (2). The procedure for synthesis of 2a is
typical. To a solution of 6a (50.8 mg, 91 mmol) in dry THF-MeOH (1 mL/
1 mL) was added a solution of sodium methoxide in MeOH (5 mol/L, 16 mL,
80 mmol), followed by stirring at room temperature for 1 hour (monitored by
TLC). The reaction was quenched by the addition of acetic acid (1 mol/L in THF,
118 mL, 118 mmol) followed by silca gel (5 mL), and concentrated under
reduced pressure. The residue was purified by dry silica gel column
chromatography (CHCl3/MeOH/H2O = 65:35:5 as an eluent) to give the
product 2a (28 mg, 79% yield) as a colorless amorphous solid after freeze–
drying from dioxane.
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23. The preliminary enzyme reactions using b-D-galactosidase from Escherichia coli
or bovine liver were also carried out, but no hydrolysis was observed (data not
shown).
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