Synthesis of three fluorescent boronic acid sensors for tumor marker Sialyl Lewis X in cancer diagnosis

Article abstract of DOI:10.1016/j.tetlet.2014.05.112

Sialyl Lewis X (sLex) is a carbohydrate that is considered not only a marker for cancer, but also an antigen associated with the malignant behavior of cancerous cells. We have synthesized three fluorescent boronic acid sensors as potential sensors for sLex. Photoinduced electron transfer by a fluorescence analyzer was used to assess sensor-sLex antigen binding. The reaction was carried out in mixed aqueous solution, and Sensor 3 was identified as showing the strongest fluorescence enhancement upon binding to sLex at 10 nM. In cell-line culture experiments, Sensor 1 was shown to label sLex expression positively for HepG2, Colo 205, and COS-7 cells, and negatively for MDA-MB-231 cells; Sensor 2 did so positively for HepG2, PLC/PRF/5, and Colo 205 cells, and negatively for MDA-MB-231 and COS7 cells; and Sensor 3 did so positively for PLC/PRF/5 and HepG2 cells, and negatively for MDA-MD-231 and COS7 cells. MTT cytotoxicity experiment results showed that the three sensors are nontoxic, and Hoechst 33258 experiments showed that no apoptosis occurred.

Full text of DOI:10.1016/j.tetlet.2014.05.112

Accepted Manuscript
Synthesis of Three Fluorescent Boronic Acid Sensors for Tumor Marker Sialyl
Lewis X in Cancer Diagnosis
May-Hua Chang, Chia-Ni Chang
PII:
S0040-4039(14)00943-5
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.05.112
TETL 44703
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 March 2014
23 May 2014
29 May 2014
Please cite this article as: Chang, M-H., Chang, C-N., Synthesis of Three Fluorescent Boronic Acid Sensors for
Tumor Marker Sialyl Lewis X in Cancer Diagnosis, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/
j.tetlet.2014.05.112
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Synthesis of Three Fluorescent Boronic Acid
Sensors for Tumor Marker Sialyl Lewis X in
Cancer Diagnosis
Leave this area blank for abstract info.
May-Hua Chang and Chia-Ni Chang
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H3C
H3C
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O
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H3C
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OH
B
OH
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OH
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HO
HO
HO
OH
H
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HO
H3C
H3C
Sensor 1
Sensor 2
Sensor 3
Sensor 1-HepG2

1
Tetrahedron Letters
journal homepage: www.els evier.com
Synthesis of Three Fluorescent Boronic Acid Sensors for Tumor Marker Sialyl Lewis X in Cancer
Diagnosis
May-Hua Chang and Chia-Ni Chang
a Industrial Technology Research Institute, Hsinchu, Taiwan, Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
Sialyl Lewis X (sLex) is a carbohydrate that is considered not only a marker for cancer, but also
an antigen associated with the malignant behavior of cancerous cells. We have synthesized three
fluorescent boronic acid sensors as potential sensors for sLex. Photoinduced electron transfer by
fluorescence analyzer was used to assess sensor-sLex antigen binding. The reaction was carried
out in mixed aqueous solution, and Sensor 3 was identified as showing the strongest
fluorescence enhancement upon binding to sLex at 10 nM. In cell-line culture experiments,
Sensor 1 was shown to label sLex expression positively for HepG2 , Colo 205, and COS-7 cells,
and negatively for MDA-MB-231 cells; Sensor 2 did so positively for HepG2, PLC/PRF/5, and
Colo 205 cells, and negatively for MDA-MB-231 and COS7 cells; and Sensor 3 did so
positively for PLC/PRF/5 and HepG2 cells, and negatively for MDA-MD-231 and COS7 cells.
MTT cytotoxicity experiments results showed that the three sensors are nontoxic, and Hoechst
33258 experiments showed that no apoptosis occurred
Received in revised form
Accepted
Available online
Keywords:
Keyword_1 Fluorescent Boronic Acid Sensors
Keyword_2 Hoechst 33258
Keyword_3 Sialyl Lewis X
Keyword_4 MTT
Keyword_5 Glycan molecules
2009 Elsevier Ltd. All rights reserved.
fluorescence-intensity increase upon binding is thought to be due
to the diminished fluorescence quenching by the transfer of
Introduction
Glycan molecules such as sialyl Lewis X (sLex; Fig 1) and sialyl
Lewis A (sLea) have often been associated with the development
of cancerous diseases. sLex is a tetrasaccharide located at the
terminus of a glycan chain and, because of this terminal
positioning, sLex epitopes are often used as clinical indicators for
serum tumor markers.1-3 Expression of these glycan molecules in
cancer cells is significantly higher than in non-malignant
epithelial cells. Over-expression of the sLex antigen has been
found to be associated with colorectal, bladder, stomach, and
gastrointestinal cancers. Increased expression of sLex has been
shown to be correlated with strong adherence of cancer cells to
E-selectin on vascular endothelial cells.4-7 Moreover, sLex is also
a key component of the carbohydrate ligands for P- and L-
selectins.8 Together, these results indicate that sLex plays an
important role in hematogenous cancer metastasis.
unbound nitrogen electrons during boronate ester formation. The
magnitude of the increase in fluorescence intensity can be
measured using a fluorescence analyzer. With this design in mind,
we synthesized three fluorescent boronic acid sensors with
appropriate bond length and structure to match the epitope of
sub-terminal tetrasaccharides on sLex. When the probe binds
strongly to the target sLex antigen through the boric acid
moieties, it will exhibit high fluorescence intensity enhancement.
The development of sensors to recognize sLex is invaluable for
the diagnosis and early detection of cancers.
Results and Discussion
Synthesis of the three fluorescent boronic acid sensors (Scheme
1) commenced by Ullmann amination10 of commercially
available 4-bromobenzaldehyde with 5 mole % copper powder in
aqueous methylamine at 100ºC. This produced an 89% yield of
4-(methylamino)benzaldehyde (1). The simplicity of the
Knoevenagel condensation of aldehyde (1) with (2,6-dimethyl-
4H-pyran-4-ylidene)malononitrile11-12 in the presence of base led
to the production of dialkylation (2) and monoalkylation
compounds (3) in yields of 73% and 26%, respectively. The free
amines (2) and (3) were then reacted separately with 2-
(bromomethyl)phenylboronic acid in the presence of potassium
carbonate in acetonitrile to produce a 94% yield of the diboronic
acid Sensor 1 and 82% yield of the monoboronic acid sensor 3.
Sensor 3 was condensed with 4-(methylamino)benzaldehyde in
the presence of piperidine in acetonitrile to produce an 89% yield
of Sensor 2.
Fig 1. Sialyl Lewis X tetrasaccharide
A typical photoinduced electron transfer (PET) sensor9 is
composed of three main components: a fluorophore, a spacer,
and a receptor. The fluorophore consists of a conjugated system
that will fluoresce when excited by UV visible light. The spacer
consists of an electron-rich group, such as nitrogen, that will
readily transfer an electron to the fluorophore, thereby quenching
fluorescence. When the receptor is positioned in the segment of
the sensor that can directly bind to the target analyte, sLex, the

2
Tetrahedron
SMMU-7721, PLF/PRF/5 and HepG2 cell lines was 7.03%,
63.35%, and 97.29%, respectively. Breast cancer cell line MDA-
MB-231 (for which CD44 is positively expressed in 90.19% of
cells according to FACS analysis). Colon cancer cell line Colo
205 carrying sialyl Lewis a and x epitopes (H-CanAg) were
purified by trichloroacetic acid precipitation and Superose 6 gel
filtration. Colo-205 expressed high levels of sLex/sLe. COS7
was used as control, because it does not express sLex, Lea, Leb,
or Ley. Cells were incubated with 10 µM of Sensor 1, Sensor 2,
or Sensor 3 in six-well plates in the dark at 4ºC. After 45 min,
NC
CN
NC
CN
CHO
CHO
(a)
(b)
H3
C
O
+
N
O
H
H3
C
NH
1
N
Br
H3
C
H
H
N
2
3
H3C
NC
NC
CN
CN
H3
C
(c)
H3
C
O
O
N
N
H
OH
B
cells were observed using
a fluorescence microscope in
HO
accordance with Weston and Wang.14 Sensor 1 showed close
integration with hepatocellular carcinoma cell-line HepG2 and
with colon cancer cell-line Colo 205, and with Cos-7; Sensor 2
showed close integration with hepatoma B cell-line PLC/PRF/5,
with HepG2, and with Colo 205; Sensor 3 showed specific
integration with PLC/PRF/5 and with HepG2 (Fig 3). MTT
cytotoxicity experiments results showed all three sensors are
nontoxic, with the cell viability of HepG2 or PLC/PRF/5 cells
after action of the three sensors remaining at nearly 100% (Fig 4).
Hoechst 33258 experiments showed no occurrence of apoptosis
(Fig 5).
H
OH
N
B
2
N
Sensor 1
H3
C
HO
H3
C
NC
CN
NC
NC
CN
CN
H3
C
O
(d)
(e)
N
H3
C
H3
C
O
O
N
N
H
OH
B
HO
OH
B
H
N
Sensor 3
Sensor 2
3
HO
H3
C
Scheme 1. (a) (i) MeNH2, Cu (89%); (b) (2,6-Dimethyl-4H-pyran-4-
ylidene)malononitrile, CH3CN (73%) (26%); (c) 2-
2
3
(bromomethyl)phenylboronic acid, K2CO3 (94%); (d) 2-
(bromomethyl)phenylboronic acid, K2CO3 (82%); (e) 4-
(methylamino)benzaldehyde (89%).
Sensor 1 Sensor 2
Sensor 3
BFI
PLC/PRF/5
HepG2
The three sensors synthesized – Sensors 1, 2, and 3 – had
emission spectra with impressive red fluorescence with a peak
climax at 617, 614, and 607 nm (400 nm excitation) in methanol,
respectively. Fluorescence binding experiments of the boronic
acid sensors with sLex were thus prepared and conducted in a
mixture of MeOH and PBS (0.1 M phosphate buffer solution,
pH 7.4) (1:1, v/v)13 The concentration of sensors was fixed at 1
µM, 0.1 µM, and 0.01 µM, while the concentration of sLex was
set at 60 µM. Based on the principles of PET, binding of the
boronic acid sensors with the target sLex should increase their
respective fluorescence intensities. As expected, we observed
increases in the fluorescence intensities of each sensor. The
profile of fluorescence intensity change was examined using a
MDA-MB
-231
Colo 205
Cos-7
fluorescence analyzer.
A favorable interaction or close
integration of the probe sensor with the sLex antigen was
observed when the fluorescence intensity of all three sensors is
greater than 200% at a sensor concentration of 1 µM. Sensor 3
was found to exhibit the strongest fluorescence enhancement
upon binding with sLex at 0.10 µM (300%) and 0.01 µM (120%)
(Fig 2).
Fig 3. Fluorescent labeling studies of PLC/PRF/5, HEPG2, MDA-MB-231,
Colo 205, and COS-7 cells by fluorescent microscopy.
5.0
4.5
S1
S2
S3
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
PLC/PRF/5
HepG2
Fig. 4 Cell viability of three sensors acting on HepG2 or PLC/PRF/5 cells
0.01
0.10
uM
1.00
Fig 2. Fluorescence binding studies by the fluorescence analyzer test.
Due to the high fluorescent enhancement of Sensor 1, Sensor 2,
and Sensor 3 upon binding with sLex, it was necessary to assess
the effect of the sensors in cell culture using sLex-expressing
cells such as hepatocellular carcinoma cell-lines (HepG2 and
PLC/PRF/5). The positive expression of sialyl Lewis X in

3
d6-DMSO) δ 159.68, 155.56, 151.65, 138.49, 129.95, 122.04,
116.16, 112.09, 111.31, 104.30, 52.64, 29.00, ESI-MS (positive
ion) m/z: 407.2 [M+H]+, HRMS (ESI) 407.1872 (407.1866
1
calculated for C26H23N4O). Monoalkylation (3) H-NMR (500
MHz, d6-DMSO) δ 7.45 (d, 2H, J=9Hz), 7.40 (d, 1H, J=16Hz),
6.95 (d, 1H, J=16Hz), 6.70 (d, 1H, J=2.5H), 6.60 (d, 1H, J=1Hz),
6.55 (d, 2H, J=8.5Hz), 6.47 (q, 1H, J=5Hz), 2.72 (d, 3H, J=5Hz),
2.41 (s, 3H), 13C-NMR (125 MHz, d6-DMSO) δ 163.81, 161.43,
156.75, 152.21, 139.16, 130.27, 122.22, 116.05, 112.23, 111.83,
105.59, 104.93, 54.07, 29.43, 19.54, ESI-MS (positive ion) m/z:
290.2 [M+H]+, HRMS (ESI) 290.1284 (290.1288 calculated for
C18H16N3O).
Fig 5 Fluorescent labeling studies and Hoechst 33258 experiments on
PLC/PRF/5 and HepG2 cells
Sensor 1
A mixture of dialkylation compound (2) (29.4 mg, 73 µmol), 2-
(bromomethyl)phenylboronic acid (128.91 mg, 0.60 mmol), and
potassium carbonate (200 mg, 1.46 mmol) in dry acetone (10
mL) was stirred at room temperature for 48 hours. The insoluble
materials were filtered off and the filtrate was evaporated in
vacuo and purification was carried out by HPLC using a
preparative column, YMC-Pack ODS-A 250 x 20 mm, and
mobile phase of acetonitrile/water (8:2), rate=8 mL/min,
retention time 16.10 min, yielded Sensor 1 in 94% (45.8 mg,
68.0 mmol). 1H-NMR (500 MHz, d6-acetone) δ (ppm): 7.68 (d,
4H, J=8Hz), 7.56 (d, 4H, J=8.5Hz), 7.32 (s, 4H), 7.26 (t, 2H,
J=8Hz), 7.18 (t, 2H, J=7Hz), 7.06 (d, 2H, J=7.5Hz), 6.93 (d, 2H,
J=16Hz), 6.79 (d, 4H, J=9Hz), 6.56 (d, 2H, J=1.5Hz), 4.86 (s,
4H), 3.09 (s, 6H), 13C-NMR (125 MHz, d6-Acetine) δ 161.03,
157.16, 152.71, 143.94, 139.59, 135.70, 130.91, 130.64, 127.01,
126.72, 124.53 ,116.81, 114.50, 113.70, 106.00, 57.44, 56.38,
39.49, ESI-MS (positive ion) m/z: 675.2 [M+H]+, 731.2
[M+4MeOH-4H2O+H]+, HRMS (ESI) 675.2964 (675.2958
calculated for C40H37B2N4O5).
Conclusion
In conclusion, we synthesized three fluorescent boronic acid
sensors capable of targeting sLex-expressing cells. Fluorescence
spectroscopy showed high intensity enhancement over a wide
range of concentrations. The results of fluorescence spectroscopy
indicated that three different types of cell lines expressing sLex
antigen showed close integration with the synthesized sensors.
Sensor 2 is the better choice for identifying both positive and
negative expression, and thus it has great potential for use in
cancer diagnosis
Acknowledgement
Financial support from the Innovation Panel of the Industrial
Technology Research Institutes is gratefully acknowledged.
Experimental
4-(Methylamino)benzaldehyde (1)
Sensor 3
A mixture of 4-bromobenzaldehyde (1.85 g, 10 mmol), 40%
aqueous methylamine solution (5.4 mL, 50 mmol), copper
powder (32 mg, 0.5 mmol), and a stirring bar was sealed in a
100-mL screw-top tube and stirred in an oil bath at 100ºC. After
a 17-hour incubation period, the reaction mixture was cooled to
room temperature and ethyl acetate (20 mL) was added to extract
the aryl amine. The organic layer was separated and the aqueous
layer was extracted using ethyl acetate (3 x 10 mL). The
combined extracts were then dried by anhydrous sodium sulfate
and the solvent was removed under reduced pressure to give a
crude product that was purified by silica gel column
chromatography EtOAc/n-hexane (1:1) to give the pure product
4-(methylamino)benzaldehyde (1.21 g, 89%, yellow crystals).
1H-NMR (500 MHz, CDCl3): δ 9.70 (s, 1H), 7.68 (d, 2H, J=8.5
Hz), 6.59 (d, 2H, J=8.5 MHz), 4.45 (s, 1H), 2.90 (s, 3H), 13C-
NMR (125 MHz, CDCl3) δ 190.29, 154.32, 132.21, 126.02,
111.34, 29.85, ESI-MS (positive ion) m/z: 136.2 [M+H]+, 158.4
[M+Na]+.
The same purification conditions were used with HPLC using a
preparative column, YMC-Pack ODS-A 250 x 20 mm, and
mobile phase, acetonitrile/water (6:4), rate=8 mL/min, retention
1
time 11.66 min, yielded Sensor 3 in 84%. H NMR (500 MHz,
d6-DMSO) δ (ppm): 8.16 (s, 2H), 7.54 (d, 1H, J=6.5Hz), 7.50 (d,
2H, J=9Hz), 7.42 (d, 1H, J=16Hz), 7.25 (t, 1H, J=6.5Hz), 7.19 (t,
1H, J=6.5 z), 7.00 (d, 1H, J=16Hz), 6.94 (d, 1H, J=7.5Hz), 6.73
(s, 2H), 6.71 (s, 1H), 6.61 (s, 1H), 4.78 (s, 2H), 3.07 (s, 3H), 2.42
(s, 3H), 13C-NMR (125 MHz, d6-DMSO) δ (ppm): 163.91,
161.28, 156.78, 151.23, 141.92, 138.70, 134.01, 130.04, 129.16,
125.90, 124.99, 122.54, 116.02, 113.06, 112.19, 105.65, 105.21,
55.75, 54.26, 19.55, ESI-MS (positive ion) m/z: 424.2 [M+H]+,
438.3 [M+MeOH-H2O+H]+, 452.3 [M+2MeOH-2H2O+H]+,
HRMS (ESI) 424.1827 (424.1831 calculated for C25H23BN3O3).
Sensor 2
A
mixture of sensor
3
(20.4 mg, 48 µmol), 4-
(methylamino)benzaldehyde (7.4 mg, 54 µmol), and piperidine
(0.1 mL) in acetonitrile (12 mL) was heated to reflux under argon
overnight. The reaction was then cooled and the mixture
evaporated to dryness under reduced pressure. Purification was
carried out using HPLC using a preparative column, YMC-Pack
ODS-A 250 x 20 mm, and mobile phase of acetonitrile/water
(8:2), rate=8 mL/min, retention time 18.26 min), yielded Sensor
(2) and (3)
A
mixture of 2,6-Dimethyl-4H-pyran-4-ylidene)malononitrile
(1.98 g, 11.4 mmol), 4-(methylamino)benzaldehyde (1) (1.25 g,
9.12 mmol), and piperidine (1 mL) in acetonitrile (50 mL) was
heated to reflux under argon overnight. The reaction was then
cooled and the insoluble material was filtered off to give
dialkylation compound 2 (1.92 g, 73%). The filtrate was then
evaporated to dryness under reduced pressure. Dichloromethane
was added to dilute the residue and filtered to give monoalylation
compound 3 (0.99 g, 26%). Dialkylation (2) 1H-NMR (500
MHz, d6-DMSO) δ 7.61 (d, 2H, J=16Hz), 7.57 (d, 4H, J=8Hz),
6.98 (d, 2H, J=16Hz), 6.64 (s, 2H), 6.58 (d, 4H, J=8.5Hz), 6.47
(dd, 2H, J=4.5, 5Hz), 2.74 (d, 6H, J=5Hz), 13C-NMR (125 MHz,
1
2 in 89% (23.1 mg, 42 µmol). H-NMR (500 MHz, d6-Acetone)
δ (ppm): 7.78-7.73 (m, 2H), 7.65 (q, 4H, J=8.5 Hz), 7.43 (s, 2H),
7.35 (t, 1H, J=8Hz), 7.27 (t, 1H, J=7Hz), 7.15 (d, 1H, J=8Hz),
7.03 (d, 1H, J=18.5Hz), 7.00 (d, 1H, J=18.5Hz), 6.85 (d, 2H,
J=8.5 Hz), 6.72 (d, 2H, J=8.5Hz), 6.66 (s, 3H), 4.90 (s, 2H), 3.13
(s, 3H), 2.08 (s, 3H), 13C-NMR (125 MHz, d6-acetone) δ (ppm):
161.21, 161.05, 157.22, 153.30, 152.72, 143.98, 140.00, 139.60,

4
Tetrahedron
135.74, 131.18, 130.92, 130.65, 127.01, 126.71, 124.55,
emission); 20X lens plates were photographed using a high-
sensitivity monochrome, 1360x1024, 6.45 µm/pixel camera
(Sony® ICX285AL CCD).
124.28, 116.84, 114.53, 113.86, 113.71, 112.98, 105.99, 105.80,
57.45, 55.19, 39.51. ESI-MS (positive ion) m/z: 541.1 [M+H]+ ,
HRMS (ESI) 541.2406 (541.2411 calculated for C33H30BN4O3).
References
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HepG2 or PLC/PRF/5 cells were seeded in 96-well plates at
1x104 cells/well, and incubated at 37°C in a 5% CO2 incubator.
After 24 h, the culture supernatant was replaced with fresh
DMEM and different concentrations of Sensor 1, 2, or 3. The
plates were incubated for 48 h. Then, 10 µL of MTT 3-(4,5-
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measured by FlexStation 3 microplate reader at a wavelength of
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1:1 methanol/PBS-treated cells as the 100% viable control and
using the following formula: (A595 of sensor-treated
samples/A595 of control)x100.
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Six-well plates were seeded with 5x105 cells per well and
incubated at 37°C and 5% CO2 for 48 h. The media was removed
and cells were washed twice with PBS. The cells were fixed with
1.5 ml of 1:1 methanol/PBS and incubated 20 min at 4°C. After
incubation, the methanol/PBS solution was removed and cells
were washed twice with PBS. Sensors 1, 2, and 3 were diluted in
1:1 methanol/PBS and added to wells at concentrations of 1.0–10
µM. One well was incubated only in methanol/PBS without a
compound, as a negative control. The plates were then incubated
in darkness at 4°C for 45 min. For nuclear staining, cells were
subsequently washed with PBS and chromatin was visualized by
exposure to Hoechst 33258 (10 µg/ml). Plates were examined
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Supplementary Material
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