Synthesis of a BODIPY library and its application to the development of live cell glucagon imaging probe

Article abstract of DOI:10.1021/ja9011657

The first BODIPY library (BD) was synthesized, and a highly selective glucagon sensor, Glucagon Yellow (BD-105), was discovered by fluorescence image-based screening method. BD library was synthesized via a Knoevenagel-type condensation reaction with 160

Full text of DOI:10.1021/ja9011657

Published on Web 07/06/2009
Synthesis of a BODIPY Library and Its Application to the
Development of Live Cell Glucagon Imaging Probe
Jun-Seok Lee,†,‡ Nam-young Kang,‡ Yun Kyung Kim,†,‡ Animesh Samanta,‡
Suihan Feng,‡,§ Hyeong Kyu Kim,‡ Marc Vendrell,⊥ Jung Hwan Park,⊥ and
Young-Tae Chang*,‡,§,⊥
Department of Chemistry, New York UniVersity, New York, New York 10003, Department of
Chemistry, MedChem Program of Life Sciences Institute, National UniVersity of Singapore,
Singapore 117543, Laboratory of Bioimaging Probe DeVelopment, Singapore Bioimaging
Consortium, Agency for Science, Technology and Research (ASTAR),
Biopolis, Singapore 138667
Received February 14, 2009; E-mail: chmcyt@nus.edu.sg
Abstract: The first BODIPY library (BD) was synthesized, and a highly selective glucagon sensor, Glucagon
Yellow (BD-105), was discovered by fluorescence image-based screening method. BD library was
synthesized via a Knoevenagel-type condensation reaction with 160 benzaldehydes and the 1,3 dimethyl-
BODIPY scaffold. Using BD compounds, a fluorescence image-based screening was performed against
three cell lines including AlphaTC1 and BetaTC6 cells which secret glucagon and insulin, respectively,
and HeLa as control cells. Out of the 160 candidate probes, one compound, Glucagon Yellow, exhibited
selective staining only in AlphaTC1 cells. The selectivity of Glucagon Yellow toward glucagon was confirmed
in vitro by comparison of its fluorescence intensity change against 19 biologically relevant analytes.
Subsequent immunostaining experiments revealed that Glucagon Yellow and the glucagon antibody
colocalized in pancreas tissue, showing a high quantitative correlation analysis by the Pearson’s coefficient
constant (Rr ) 0.950). These results demonstrated the potential application of Glucagon Yellow as a
glucagon imaging agent in live cells and tissues.
motrypsin,10 human serum albumin (HSA),11 glutathione,12 and
myotube cell state.13 On the basis of the successful experience
Introduction
Fluorescent small molecules have been widely used for
sensors and bioimaging probes.1 Most fluorescent probes have
been developed on a case-by-case basis by connecting a
recognition motif to a fluorophore.2 While there are many
successful cases reported, the conventional approach usually
limits the scope of the probes to known binding motifs or
fluorophores. As an alternative approach, our research group
has pursued a diversity-oriented fluorescence library approach
(DOFLA) and demonstrated the generality of this approach by
developing sensors and bioimaging agents for various targets,
including DNA,3 RNA,4,5 ꢀ-amyloid,6,7 GTP,8 heparin,9 chy-
with the previous fluorophore library, we are expanding this
DOFLA concept to a superior fluorophore, 4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene (BODIPY) to develop high performance
imaging probes.
BODIPY has outstanding photophysical properties as a
fluorescent scaffold, such as high photostability, high fluorescent
quantum yield, high extinction coefficient, and narrow emission
bandwidth.14 The chemical modification of the BODIPY scaf-
fold has been well studied.15 However, a systematic construction
of a library based on BODIPY has not been reported yet, maybe
(7) Li, Q.; Min, J.; Ahn, Y. H.; Namm, J.; Kim, E. M.; Lui, R.; Kim,
H. Y.; Ji, Y.; Wu, H.; Wisniewski, T.; Chang, Y. T. ChemBioChem
2007, 8, 1679–1687.
† Department of Chemistry, New York University.
‡ Department of Chemistry, National University of Singapore.
§ MedChem Program of Life Sciences Institute, National University of
Singapore.
(8) Wang, S.; Chang, Y. T. J. Am. Chem. Soc. 2006, 128, 10380–10381.
(9) Wang, S.; Chang, Y. T. Chem. Commun. 2008, 1173–1175.
(10) Wang, S.; Kim, Y. K.; Chang, Y. T. J. Comb. Chem. 2008, 10, 460–
465.
⊥ Laboratory of Bioimaging Probe Development, Singapore Bioimaging
Consortium, Agency for Science, Technology and Research (ASTAR).
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1517–1549.
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2007, 9, 1079–1083. (b) Ahn, Y. H.; Lee, J. S.; Chang, Y. T. J. Comb.
Chem. 2008, 10, 376–380.
(2) (a) deSilva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV.
1997, 97, 1515–1566. (b) Martinez-Manez, R.; Sancenon, F. Chem.
ReV. 2003, 103, 4419–4476.
(12) Ahn, Y. H.; Lee, J. S.; Chang, Y. T. J. Am. Chem. Soc. 2007, 129,
4510–4511.
(13) Wagner, B. K.; Carrinski, H. A.; Ahn, Y. H.; Kim, Y. K.; Gilbert,
T. J.; Fomina, D. A.; Schreiber, S. L.; Chang, Y. T.; Clemons, P. A.
J. Am. Chem. Soc. 2008, 130, 4208–4209.
(3) Lee, J. W.; Jung, M.; Rosania, G. R.; Chang, Y. T. Chem. Commun.
2003, 1852–1853.
(4) Li, Q.; Chang, Y. T. Nat. Protoc. 2006, 1, 2922–2932.
(5) Li, Q.; Kim, Y.; Namm, J.; Kulkarni, A.; Rosania, G. R.; Ahn, Y. H.;
Chang, Y. T. Chem. Biol. 2006, 13, 615–623.
(14) Karolin, J.; Johansson, L. B. A.; Strandberg, L.; Ny, T. J. Am. Chem.
Soc. 1994, 116, 7801–7806.
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Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem., Int. Ed. 2008,
47, 1184–1201.
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Y. T. Angew. Chem., Int. Ed. 2004, 43, 6331–6335.
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Scheme 1. Synthesis of BD Librarya
Although there have been extensive biological studies about
glucagon secretion and its related diseases such as diabetes, there
are no small-molecule fluorescent probes to visualize glucagon to
date. Therefore, the development of fluorescent probes that could
quantify the concentration of glucagon in live cells or tissues would
be of great importance.
Results and Discussion
Design and Synthesis of a BODIPY Library. In order to
introduce systematic structural diversity on the BODIPY scaf-
fold, we designed a synthetic scheme to append different styryl
motifs to the BODIPY fluorophore. Styryl-derivatized BODIPY
compounds are known to display red-shifted fluorescence
emissions due to the extended π-conjugation,19 and their emis-
sion wavelengths vary according to the styryl domain. Since
hundreds of aldehyde building blocks are commercially avail-
able, styryl-containing BODIPY compounds constitute a feasible
target for combinatorial library synthesis. While the syntheses
of both monostyryl and distyryl compounds have been reported
from symmetric tetramethyl-BODIPY intermediates, the isola-
tion of the two possible condensation products would make
difficult the construction of a library.19 Therefore, in order to
make easier such complex purification procedures, we synthe-
sized 1,3-dimethyl-BODIPY scaffold (3), which can only lead
to the monostyryl products. Compound 3 was prepared by
condensation of pyrrole-2-carboxylaldehyde and 2,4-dimeth-
ylpyrrole at low temperature, followed by the addition of
BF3 ·OEt2.20 Second, the monostyryl-containing BODIPY library
(named BD) was constructed using 238 aromatic aldehydes via
Knoevenagel condensation under microwave irradiation, purify-
a Reagents and conditions: (a) POCl3, dichloromethane, -5 °C for 3 h,
then 3 h at rt; (b) BF3 ·OEt2, DIEA, rt for 3 h (overall yield: 51%); (c)
R-CHO (160 aromatic aldehydes, see Table S1, Supporting Information),
pyrrolidine, AcOH, EtOH, microwave irradiation, 2-15 min.
due to the synthetic challenges.16 Here, we report the synthesis
of the first BODIPY-based library, and the subsequent discovery
of a glucagon sensor.
Glucagon is an important hormone peptide involved in carbo-
hydrate metabolism. It is produced by alpha cells in the pancreas
and maintains the level of glucose in the blood by effects opposite
to those of insulin.17 Glucagon release in alpha cells is normally
stimulated as the concentration of glucose in the blood decreases,
a response that is progressively diminished in type 1 diabetes.18
Figure 1. (A) Fluorescence microscope images of BD-105 in three different cell lines. Cells were stained with the compound at 500 nM for 24 h. Upper
row corresponds to the bright-field images, and the lower row illustrates the fluorescence images (TRITC channel: excitation 540 nm, emission 580 nm).
Scale bar: 20 µm (B) In Vitro fluorescence responses of BD-105 (1 µM) toward glucagon. Spectra obtained in 10 mM HEPES (pH 7.4) with excitation at
530 nm.
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BODIPY and Live Cell Glucagon Imaging Probes
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Figure 2. Fluorescence response of BD-105 toward a series of analytes. The intensities of BD-105 in the presence of each analyte (Fa) were compared with
fluorescence intensities of BD-105 (Fd) in 10 mM HEPES buffer (pH 7.4). Bars represent the ratio of Fa/Fd. The concentration of small-molecule analytes
(blue background) ranged from 40 µM to 5 mM, and that of macromolecule analytes (green background) from 0.125 mg/mL to 1 mg/mL.
ing the final products by semi-preparative RP-HPLC (Scheme
1). After HPLC-MS characterization, 160 compounds were
collected for further studies (average purity 96.5% at 250 nm,
Table S2, Supporting Information).
Spectroscopic Properties. The spectroscopic properties of the
BD compounds are summarized in Table S2 Supporting
Information. All library compounds exhibited red-shifted fluo-
rescence emissions compared to 3 (λem ) 512 nm). Their
spectroscopic properties are diverse, with maximum absorption
wavelengths ranging from 525 to 605 nm, and maximum
emission wavelengths from 560 to 730 nm. BD compounds
displayed relatively low fluorescence quantum yields compared
to 3, with an average value of 0.32, which maybe due to their
higher structural flexibility. These findings suggest that many
of the BD compounds could be used not only as fluorescent
quenching sensors but also as fluorescent turn-on sensors.
Image-Based Screening in Live Cells. Based on our previous
experience,5 the successful discovery of novel imaging probes
is highly dependent on both the chemical diversity of the
Figure 3. Fractional saturation curve of Glucagon Yellow vs glucagon.
Various concentrations of Glucagon Yellow were incubated with 2 µM
glucagon for 30 min at rt, followed by the measurement of the fluorescence
fluorescence library and the screening methodology. Regarding
the screening methods, two opposite approaches can be used:
target-to-system and system-to-target. Target-to-system ap-
proaches utilize high-throughput in Vitro screening techniques
emission. Experimental KD ) 13.3 µM.
to find probes, which undergo fluorescence property changes
upon interaction with a certain analyte. After a primary selection,
successful candidates are further validated for biologically
relevant applications.7,12 On the other hand, system-to-target
approaches initially screen collections of fluorophores in
complex biological systems, and further investigate the origin
of their fluorescence response in a forward chemical genetics
manner. For example, imaging agents of DNA and RNA were
discovered after in Vitro screening a series of nucleus- or
nucleolus-staining primary hits against DNA and RNA,
respectively.3,5 Because the intrinsic cellular localization of the
fluorophores may allow their specific interaction with some
biomolecules, this approach can minimize the number of false
positive hits and accelerate the overall probe discovery process.13
Cell permeability and reasonable fluorescent quantum yields are
critical requirements for the application of system-wide cell image-
based screenings. Nonpermeable probes or significantly dimmed
fluorophores are not practical tools, as they need a large amount
of compound, which may induce some cytotoxic effects. Having
determined a reasonable fluorescence quantum yield for the BD
compounds, we tested their cell permeability in 3T3-L1 and HeLa
cells. Most of the BD library members showed good cell perme-
ability (data not shown), as many previously reported BODIPY
probes,21 and the BD library was proven to fulfill the requirements
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Lee et al.
Figure 4. Normalized fluorescence excitation and emission spectrum of Glucagon Yellow (10 µM), before and after binding to glucagon. Before addition
of glucagon: absorption λmax 550 nm, emission λmax 568 nm; upon binding to glucagon: absorption λmax 553 nm, emission λmax 570 nm. All experiments were
performed in 10 mM HEPES buffer (pH 7.4).
Figure 5. Fluorescence colocalization images of Glucagon Yellow in AlphaTC1 cells. Fluorescence microscope images of Glucagon Yellow (a: TRITC
channel), glucagon antibody (b: Cy5 channel), and merged images (c). Colocalization scatter plot (d: Pearson’s coefficient 0.896). Scale bar 20 µm.
for cell-based fluorescence image evaluation. BD compounds were
screened toward three distinct cell lines as hormone peptide sensors.
We selected AlphaTC1, BetaTC6, which respectively secrete
glucagon and insulin,22 and HeLa cells as a control. All the
compounds were tested by image-based high-throughput screening,
and fluorescence images were taken at different incubation times.
Bright field images were also taken to ensure cell viability. Notably,
after 24 h incubation BD-105 exhibited a selective and high
fluorescent intensity in AlphaTC1, which is a glucagon secreting
cell line, compared to BetaTC6 or HeLa cells (Figure 1 A),
indicating its potential selective fluorescence increase upon interac-
tion with glucagon.
In Vitro Fluorescence Response. After in Vitro experiments
indicated a strong fluorescence increase (up to 17-fold) of BD-
105 upon treatment with glucagon (Figure 1-B), we proceeded to
evaluate its selectivity by screening the compound against a set of
18 biologically relevant analytes. BD-105 displayed an impressive
selectivity toward glucagon compared to serum albumins (both
BSA and HSA, common nonselective hydrophobic fluorescence
increasing proteins, Figure 2) and insulin, the counter-regulatory
hormone (Figure S3, Supporting Information). On the basis of its
selectivity pattern and visual yellow fluorescence color, we decided
to name BD-105 as “Glucagon Yellow”.
Yellow were titrated with glucagon peptide. Fractional
saturation curve of Glucagon Yellow with glucagon revealed
a dissociation constant (KD) of 13.3 µM (Figure 3). Glucagon
Yellow (1 µM) displayed a weak fluorescence emission (QY
) 0.012) in 10 mM HEPES buffer (pH 7.4), with an
absorption maximum at 550 nm and an emission maximum
of 568 nm (Figure 4). Upon addition of glucagon (200 µM)
to the solution, a marked increase of the fluorescence intensity
was observed (QY ) 0.151), with a slight red-shifted
absorption (553 nm) and emission (570 nm) maxima.
Immuno-Staining of Glucagon in AlphaTC1 cell and Pan-
creatic Tissue. To validate the biological relevance of Glu-
cagon Yellow as a glucagon-imaging agent, we further
compared the staining pattern of the probe with a fluores-
cently labeled glucagon antibody. Glucagon Yellow proved
to colocalize with a glucagon antibody, both in the AlphaTC1
cells and in mouse pancreas tissue sections, with a high
linearity in their colocalization scatter plot (Figures 5 and
6). The quantitative colocalization analysis demonstrated that
the fluorescence signals from Glucagon Yellow and the
glucagon antibody showed a positive correlation with a high
Pearson’s coefficient (Pearson’s coefficient ranges from -1
to 1. Values close to 1 indicate a reliable colocalization)
(Rr ) 0.950) from the pancreas tissue.23 Additionally, the
staining pattern of Glucagon Yellow differed from that of
insulin antibody image, demonstrating the clear selectivity
Fluorescence Titration and Spectroscopic Change by Bind-
ing to Glucagon. To further investigate the binding properties
of Glucagon Yellow, various concentrations of Glucagon
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BODIPY and Live Cell Glucagon Imaging Probes
A R T I C L E S
Figure 6. Fluorescence colocalization images of Glucagon Yellow in pancreas tissue. Fluorescence microscope images of Glucagon Yellow (a, e: TRITC
channel), glucagon antibody (b: Cy5 channel), insulin antibody (f: Cy5 channel) and merged images (c for a plus b, g for e plus f). Colocalization scatter
plot (d: Pearson’s coefficient 0.950, h: Pearson’s coefficient 0.681). Scale bar 20 µm.
purification. Aldehyde building blocks were purchased from Ald-
rich, Acros, and Maybridge Inc. Normal phase chromatography of
the intermediates was performed using Merck Silica Gel 60 (particle
size: 0.040-0.063 mm, 230-400 mesh ASTM), and the solvents
used for the spectroscopy experiments were of spectrophotometric
grade. All library compounds were purified in a semipreparative
Gilson RP-HPLC using a C18 column (100 mm × 21.2 mm, Axia
column from Phenomenex Inc.). LC-MS characterization was
performed on a LC-MS-IT-TOF Prominence Shimadzu Technology,
using a DAD (SPD-M20A) detector, and a C18 column (20 mm ×
of this probe to glucagon in both AlphaTC1 cells and
pancreas tissue (Figure 6g).
Conclusion
We have synthesized the first BODIPY library (BD library)
as potential bioimaging probe toolbox, and discovered the first
live cell/tissue glucagon fluorescent probe. One hundred and
sixty BD compounds were prepared by condensation with a
series of aldehydes, and a diverse range of the spectroscopic
properties for the final compounds was observed. By means of
a fluorescence image-based screening against three cell-lines,
we identified a unique imaging probe, Glucagon Yellow,
capable to selectively stain AlphaTC1 cells. Further experiments
indicated a selective fluorescence emission increase upon
interaction of glucagon. Immunostaining experiments demon-
strated that Glucagon Yellow colocalizes selectively with
glucagon antibody not only in AlphaTC1 cells but also in
pancreas tissue. This is the first example of a small-molecule
fluorescent probe that can be used to visualize glucagon in live
cells, representing a useful imaging probe for biological studies
of glucagon secretion and glucagon-related diseases.
˚
4.0 mm, 100 A, Phenomenex Inc.), with 4 min elution using a
gradient solution of CH3CN-H2O (containing 0.1% TFA) and an
electrospray ionization source. 1H and 13C NMR spectra were
recorded on a Bruker Advance 300 NMR spectrometer.
1,3-Dimethyl 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (1,3-
Dimethyl-BODIPY, 3). Pyrrole 2-carboxyaldehyde (300 mg, 3.15
mmol) was dissolved in absolute dichloromethane (4 mL) and
cooled down to -5 °C under nitrogen atmosphere. 2,4-
dimethylpyrrole (324.6 µL, 3.15 mmol) was added to the reaction
mixture and stirred for 3 min, followed by slow dropwise
addition of POCl3 (288.3 µL, 3.15 mmol). The reaction mixture
was stirred at -5 °C for 3 h, then another 3 h at rt. Once the
starting materials were consumed following TLC monitoring,
1.5 mL (9.45 mmol) of diisopropylethylamine (DIEA) and 1.2
mL (9.45 mmol) of BF3OEt2 were added. After 3 h, the reaction
mixture was washed with H2O (×3) and dried over Na2SO4. The
solvent was evaporated, and the residue was purified by silica
gel column chromatography (hexane/EtOAc ) 5:1) to yield 227.2
Experimental Section
Materials and Methods. All the materials were obtained from
commercial suppliers (Acros and Aldrich) and used without further
1
mg (1.03 mmol, 32.8%) of 3 as a red solid product. H NMR
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(300 MHz, CDCl3) δ 2.28 (s, 3H), 2.59 (s, 3H), 6.16 (s, 1H),
6.43 (s, 1H), 6.93 (s, 1H), 7.20 (s, 1H), 7.64(s, 1H). ESI-MS
m/z (M+) calc: 220.10, found: 243.10 (M + Na).
General Procedure for the Aldehyde Condensation Reac-
tion. Method 1 (for aromatic aldehydes containing phenol moieties):
3 (15 mg, 68 µmol) and aldehyde (68 µmol, 1 equiv) (Table S1)
were dissolved in absolute EtOH, with 10 equiv of pyrrolidine (48
µL, 680 µmol) and 10 equiv of AcOH (35 µL, 680 µmol). The
condensation reaction was performed by using consecutive 1 min-
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Chem. 2005, 280, 29053–29059. (c) Verdoes, M.; et al. Chem. Biol.
2006, 13, 1217–1226. (d) Domaille, D. W.; Que, E. L.; Chang, C. J.
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step microwave irradiation (700 W). After every step, the reaction
mixture was cooled down to rt and then monitored by TLC. The
reactions were finished between 5 to 20 min. The resulting crude
mixtures were concentrated under vacuum, and purified by semi-
preparative RP-HPLC. Combined purification fractions were dried
using a GeneVac evaporator. Method 2 (for rest of aromatic
aldehydes): 3 (15 mg, 68 µmol) and aldehyde (68 µmol, 1 equiv)
(Table S1, Supporting Information) were dissolved in absolute
EtOH, with 10 equiv of pyrrolidine (48 µL).The condensation
reaction was performed by using consecutive 1 min-step microwave
irradiation (700 W). After every step, the reaction mixture was
cooled down to rt, and then monitored by TLC. The reactions were
finished between 5 to 20 min. The resulting crude mixtures were
concentrated under vacuum and purified by semipreparative RP-
HPLC. Combined purification fractions were dried using a GeneVac
evaporator.
software. Glucagon (2 µM) solution was titrated by different
concentrations of Glucagon Yellow (0.5-100 µM), and the
fluorescence intensities were measured at 568 nm with (excitation:
520 nm). The bound fraction (X) of Glucagon Yellow at each
concentration was determined using the eq 2
Fc - Fo
Fsat - Fo
X )
(2)
where Fc and Fo are the fluorescence intensities of a given
concentration of Glucagon Yellow with and without glucagon,
respectively. Fsat is the fluorescence intensity at the same concentra-
tion of Glucagon Yellow when fully bound. Fsat was determined
by fluorescence titration at each concentration with a series
concentration of glucagon. The results were plotted according to
nonlinear fitting curve eq 3
Characterization of BD-Library. All the library compounds
were characterized by LC-MS (See Table S2, Supporting Informa-
tion).
F ) Fo + (Fsat - Fo)([GY])/(KD + [GY])
(3)
Characterization of Glucagon Yellow (BD-105): 1H NMR (300
MHz, DMSO-D6) δ 2.35 (s, 3H) 6.51 (dd, J ) 1.80, 3.72 Hz, 1H)
6.82 (m, 1H) 7.02-7.14 (m, 4H) 7.26 (t, J ) 7.71 Hz, 1H) 7.37
(d, J ) 16.4 Hz, 1H, C)CH trans) 7.70 (d, J ) 16.3 Hz, 1H, C)CH
trans) 7.71 (s, 1H) 7.80 (s, 1H) 9.66 (s, 1H), 13C NMR (75.5 MHz,
DMSO-D6) δ 11.56, 113.58, 116.99, 117.66, 117.97, 118.16,
119.55, 125.32, 127.04, 130.62, 133.34, 137.18, 137.72, 139.16,
141.20, 145.85, 158.25, 158.48, ESI-MS (m/z) calcd: 324.10 found
325.10 (M + H).
Quantum Yield Measurements. Quantum yields were calcu-
lated by measuring the integrated emission area of the fluorescent
spectra and comparing that value to the area measured for
rhodamine B in EtOH when excited at 500 nm (Φrho-B ) 0.70).
Quantum yields for the BD library were then calculated using eq
1, where F represents the area of fluorescent emission, n is reflective
index of the solvent, and Abs is absorbance at excitation wavelength
selected for standards and samples. Emission was integrated
between 530 to 700 nm.
where KD is the dissociation constant, [GY] is the concentration of
Glucagon Yellow.
Cell Culture. 3T3-L1 and HELA cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM, GIBCO) supplemented with 10%
fetal bovine serum (FBS) and antibiotics (100 µg/mL penicillin/
streptomycin mixture) in a humidified atmosphere at 37 °C with
5% CO2. AlphaTC1 Clone 9 (ATCC) were grown in Dulbecco’s
Modified Eagle’s Medium (DMEM, GIBCO) with 4 mM L-
glutamine, 3.0 g/L glucose, 1.5 g/L sodium bicarbonate, and 10%
heat-inactivated dialyzed fetal bovine serum in a humidified
atmosphere at 37 °C with 5% CO2. Beta-TC-6 (ATCC) cells were
grown in Dulbecco’s Modified Eagle’s medium (DMEM, GIBCO)
with 15% heat-inactivated fetal bovine serum in a humidified
atmosphere at 37 °C with 5% CO2.
Immunohistochemistry (Frozen Mouse Pancreas). Mouse
pancreas was cut into smaller pieces (∼5 mm long) with a razor
blade, and 10 µm sections were cut on a MICROM/HM520.
Sections were used within one week of cutting. Before staining,
the sections were fixed with cold acetone for 10 min. Immunohis-
tochemistry images of frozen sections with the anti-glucagon
monoclonal antibody and Glucagon Yellow were obtained from
NIKON T1 fluorescence microscopy (540/580 nm filter-TRITC
channel for Glucagon Yellow and Cy5 filter (650/680 nm) for
glucagon antibody visualization).
Fsample
ηsample Absreference
sample
flu
reference
fl
Φ
) Φ
(1)
(
)(
)(
)
Freference ηreference Abssample
In Vitro Fluorescence Screening. BD-105 (10 µM) was
evaluated in 10 mM HEPES, pH 7.4. Fluorescence intensities were
measured using a Spectra Max Gemini XSF plate reader in 384-
well format. Excitation was provided at 530 nm, and emission was
obtained starting from 560 to 630 nm. All the analytes were tested
at four serial concentrations: macromolecule analytes ranged from
0.125 mg/mL to 1 mg/mL (BSA, HSA, cytochrome C (bovine
heart), glucagon, histone, IgG(bovine), IgG(human), insulin, trans-
ferrin(apo), transferrin(holo), ubiquitin), and concentrations of small-
molecule analytes varied from 40 µM to 1 mM (DTT, GSH, GSSG,
H2O2, NAD, NADH, ascorbic acid).
Acknowledgment. We gratefully acknowledge National Uni-
versity of Singapore (NUS) for the financial support (Young
Investigator Award: R-143-000-353-123). J.S.L. was supported by
a Korea Research Foundation Grant funded by the Korean govern-
ment (MOEHRD, Basic Research Promotion Fund: KRF-2005-
C00088).
Supporting Information Available: Complete ref 21c; HPLC
data and chemical structures for the 160 BD compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Determination of the Dissociation Constant for Glucagon
Yellow to Glucagons. The fluorescent emission spectra of glucagon
with various concentrations of Glucagon Yellow were measured
on a SpectraMax M2 plate reader. The fluorescent titration curve
was fitted to the standard equation using Graphpad Prism v5
JA9011657
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10082 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009