Accelerating fluorescent sensor discovery: Unbiased screening of a diversity-oriented BODIPY library

Article abstract of DOI:10.1039/c0cc04495d

Herein, we report the first systematic and unbiased evaluation of the BODIPY fluorophore library against a wide panel of biologically relevant molecules, and discoveries of 2 novel fluorescent probes for BSA and dopamine.

Full text of DOI:10.1039/c0cc04495d

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COMMUNICATION
Accelerating fluorescent sensor discovery: unbiased screening
of a diversity-oriented BODIPY libraryw
ab
a
Jun-Seok Lee,z Hyeong Kyu Kim,z Suihan Feng,a Marc Vendrellc and
Young-Tae Chang*ac
Received 19th October 2010, Accepted 24th November 2010
DOI: 10.1039/c0cc04495d
Herein, we report the first systematic and unbiased evaluation
of the BODIPY fluorophore library against a wide panel
of biologically relevant molecules, and discoveries of 2 novel
fluorescent probes for BSA and dopamine.
binding partners. Our group previously reported the com-
binatorial synthesis of 160 monostyryl-BODIPY dyes, which
exhibited an average fluorescent quantum yield around 0.32.8
In view of the potential of mono-styryl BODIPY dyes as
both fluorescent turn-on and turn-off probes, we extended
the mono-styryl BODIPY library (BD) using 505 aldehyde
building blocks and the asymmetric 1,3 dimethyl-BODIPY
scaffold in a Knoevenagel condensation reaction (Scheme 1).
A total of 317 BD dyes with purities over 95% were collected
for further evaluation, and showed broad absorbance and
emission profiles (labs: 525–616 nm, lflu: 540–656 nm), and
an averaged quantum yield of 0.49 (for chemical structures
and full spectroscopic characterization, see Tables S1 and S2
in Supporting Informationw).
High-throughput unbiased screening strategies have been
successfully used in drug discovery,9 but they are still at an
early stage in fluorescent sensor development. Unbiased sensor
discovery screenings must enclose a number of biomolecule
targets that embody broad biological events/interests and
resemble the conditions in which the sensor will be eventually
applied. Therefore, a total of 94 biomolecules in the range of
their endogenous or effective concentrations were systematically
combined with the BD compounds, and the resulting fluores-
cence pattern was examined. HEPES buffer was chosen as the
vehicle since it can simulate better the intracellular conditions,
and the 94 biomolecules were categorized in 10 sets: pH and
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes
have attracted much attention in fluorescence imaging due to
their outstanding photophysical properties (e.g. high extinc-
tion coefficient and photostability, narrow bandwidth, etc).1 In
particular, high fluorescence quantum yield and environmentally
insensitive character made BODIPY fluorophore one of the
popular labelling agents to visualize biomolecules,2 including
ceramides,3 peptides,4 and various metabolites.5 On the other
hand, there are limited numbers of BODIPY based chemo-
sensors. These sensors were generally designed by connecting
the fluorophore to recognition motifs (e.g. ion chelators) and
exploit the photon electron transfer (PET) mechanism to
monitor the binding process by fluorescence changes.6 Despite
the usefulness, the scope of these approaches is restricted to a
few number of recognition events. In view of the collective
limitations to design new binding modes and turn them into a
fluorescence response, unbiased high-throughput screenings
can be a practical strategy to accelerate the development of
novel fluorescent probes.7 Herein, we report the first systematic
and unbiased analysis of the BODIPY fluorophores against a
wide panel of biomolecules. The screening of a 317-member
library against 94 biomolecules led to the discovery of two
novel BODIPY probes for BSA and dopamine.
Fluorescent sensors must ideally display a moderate quantum
yield that allows the observation of their fluorescence intensity
increase/decrease upon interaction with the corresponding
a Department of Chemistry, National University of Singapore,
3 Science drive 3 Singapore. E-mail: chmcyt@nus.edu.sg;
Web: http://ytchang.science.nus.edu.sg; Fax: (+65) 6779-1691
b Life/Health Division, Integrated Omics Center,
Korea Institute of Science and Technology (KIST), Korea
c Laboratory of Bioimaging Probe Development,
Singapore Bioimaging Consortium, Agency for Science,
Technology and Research (A*STAR), 11 Biopolis way,
Singapore 138667
w Electronic supplementary information (ESI) available: detail charac-
terizations of BODIPY library are available. See DOI: 10.1039/
c0cc04495d
z These authors contributed equally to this work.
Scheme 1 Synthesis of the BD library. Reagents and conditions:
(i) POCl3, DCM, ꢀ5 1C for 3 h, then r.t. for 3 h; (ii) BF3ꢁOEt2,
DIEA, 3h, r.t.; (iii) pyrrolidine/AcOH, R–CHO, ACN, microwave
irradiation, (700 W).
c
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Chem. Commun., 2011, 47, 2339–2341 2339

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pockets.15 However, the design of ligands that are species-
selective is still challenging,16 since serum albumins share a
high sequence homology that makes them undistinguishable
even by crystallographic methods.14,17 Few BSA-selective
fluorescent sensors are available, and to the best of our
knowledge the most sensitive BSA probe (FA: 2-(6-diethyl-
aminobenzo[b]furan-2-yl)-3-hydroxychromone) shows a limited
6-fold fluorescence increase.18 BD-101 underwent an exceptional
212-fold fluorescence increase with
a dissociation con-
stant (KD) of 5.24 mM when binding to BSA (Fig. 2a, S2w),
and the unbiased screening asserted its excellent selectivity
profile with a negligible response against all other tested
biomolecules (Fig. 2b). Furthermore, BD-101 showed a
clear selectivity for BSA when comparing 5 different serum
albumins (bovine, human, rat, porcine, and sheep: Fig. S1w),
being the most responsive BSA fluorescent sensor reported
to date.
Fig.
1 Heatmap plot of the fluorescence pattern of 317 BD
compounds against 94 biomolecules. Green and red colors indicate a
max
fold change increase and decrease respectively at lem
compound (Fold = Fanalyte/Fvehicle).
of each
viscous solutions, nucleotides and nucleosides, genetic macro-
molecules, peptides, proteins, metal cations, oxidation–reduction
related molecules, pesticides, and other miscellaneous
analytes (see Table S3 for detailsw). The fluorescence spectra
of every compound was recorded at four serial analyte
concentrations (94 analytes ꢂ 4 concentrations ꢂ 317 BD
compounds = 119 192 data points), and the quality of
the screening data was cross-validated by comparing dose-
response patterns for a given biomolecule so that the number
of false positive hits could be minimized.10 The overall fluores-
cence responses of the 317 BD compounds were combined
using a heatmap plot (Fig. 1). Heatmap plot represents fold
change values of fluorescence emission as two color codes
(green for turn-on and red for turn-off), which facilitated the
deduction of the general behaviour of BD compounds as
fluorescent probes: (1) derivatives containing a p-aminobenzyl
or a heterocyclic group showed a very high fluorescence
increase at low pH (Table S5w); (2) proteins showed the
highest response amongst all analyte classes, and (3) many
BD compounds showed a fluorescence intensity decrease in the
presence of negatively charged biomolecules, such as genetic
macromolecules (DNA, RNA) and nucleotides/nucleosides.
In addition to characterizing the general fluorescence profile
of the BD library, the analysis of the unbiased screening data
identified two compounds (BD-101 and BD-187) with an out-
standing turn-on/off responses (BSA and dopamine, respectively),
and we further studied their application as fluorescent probes.
BD-101 showed a remarkable fluorescence turn-on effect upon
binding to bovine serum albumin (BSA). Serum albumins are
the major constituent of blood plasma,11 and play a major role
in transporting diverse endogenous biomolecules and drugs.11,12
Several studies have been aimed at describing BSA drug
Fig. 2 (a) Fluorescence emission spectra of BD-101 in the presence of
serial concentrations of BSA (lexcitation: 530 nm); (b) Fluorescence
response profile of BD-101 (10 mM) upon unbiased screening
(detail decoding table is in Table S4w), and BSA was marked with a
red arrow.
13
binding sites and their structural characterization,14 as well
as developing fluorescence probes for the different binding
c
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The diverse quantum yields of the BD library enabled the
discovery of compounds behaving as fluorescent turn-off
probes. BD-187, BD-404 and BD-405 displayed a significant
fluorescence quenching upon interaction with dopamine, a
catecholamine neurotransmitter.19 These three BD compounds
were the only library members containing a boronic acid
group, which can potentially interact with the catechol moiety
of dopamine.20 Interestingly, the fluorescence quenching effect
showed a strong dependency on the position of the boronic
acid. Whereas the ortho-substituted analogue, BD-405,
exhibited the weakest quenching effect, the para-substituted
compound BD-187 showed an exceptional 10-fold fluores-
cence emission decrease with a dissociation constant (KD) of
0.49 mM (Fig. 3, S3w). BD-187 quenching followed a linear
correlation with dopamine concentration up to 1 mM, which
exceeded the sensitivity of the IDA and dopamine sensors
reported so far.21
In summary, we have systematically examined the fluores-
cence response of a diversity-oriented BODIPY library (BD)
using an unbiased in vitro screening strategy. Based on the
quantum yield changes against a variety of biomolecules, we
could infer general trends of the BODIPY fluorescence response
pattern, and discover two novel fluorescent probes: BD-101
behaved as a turn-on sensor for BSA, while BD-187 showed a
remarkable fluorescence quenching upon interaction with
dopamine. With this first unbiased screening of the fluores-
cence pattern of the BODIPY structure, we demonstrated the
power and broad applicability of this approach for accelerating
fluorescent sensor discovery, without prior implementation of
a pre-established binding motif in the fluorophore structure.
This work was supported by an intramural funding from
A*STAR Biomedical Research Council and National Univer-
sity of Singapore (YIA:R-143-000-353-123). J.-S.Lee thanks
for the POSCO TJ Park Bessemer Science Fellowship.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2339–2341 2341