MegaStokes BODIPY-triazoles as environmentally sensitive turn-on fluorescent dyes

Article abstract of DOI:10.1039/c3sc22166k

A novel class of triazole-derivatized BODIPY compounds have been synthesized on solid-phase by employing mild reaction conditions based on the copper-catalyzed azide-alkyne cycloaddition. The resulting BODIPY-triazoles exhibited MegaStokes shifts (up to 160 nm) and remarkable environmentally sensitive quantum yield increments that asserted their potential as turn-on fluorescent sensors. Out of a library of 120 compounds, we identified BDC-9 as a fluorescent chemosensor with high sensitivity and remarkable species-selectivity towards human serum albumin. These results validate MegaStokes BODIPY dyes as new fluorophores for the development of environmentally sensitive fluorescent probes.

Full text of DOI:10.1039/c3sc22166k

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MegaStokes BODIPY-triazoles as environmentally
sensitive turn-on fluorescent dyes†
Cite this: DOI: 10.1039/c3sc22166k
a
a
a
cd
Jun Cheng Er,ab Mui Kee Tang, Chee Geng Chia, Huimin Liew, Marc Vendrell
and Young-Tae Chang
*
abd
*
A novel class of triazole-derivatized BODIPY compounds have been synthesized on solid-phase by
employing mild reaction conditions based on the copper-catalyzed azide–alkyne cycloaddition. The
resulting BODIPY-triazoles exhibited MegaStokes shifts (up to 160 nm) and remarkable environmentally
sensitive quantum yield increments that asserted their potential as turn-on fluorescent sensors. Out of a
library of 120 compounds, we identified BDC-9 as a fluorescent chemosensor with high sensitivity and
remarkable species-selectivity towards human serum albumin. These results validate MegaStokes
BODIPY dyes as new fluorophores for the development of environmentally sensitive fluorescent probes.
Received 6th December 2012
Accepted 20th February 2013
DOI: 10.1039/c3sc22166k
www.rsc.org/chemicalscience
the corresponding acetyl (BDCAC) and chloroacetyl (BDCCA)
derivatives (Scheme 1). Moreover, the library was synthesized
1 Introduction
The development of uorescent and molecular imaging probes
based on the 4,4-diuoro-4-bora-3a,4a-diaza-s-indacene (BOD-
IPY) scaffold has been widely exploited due to its outstanding
uorescent properties (e.g. high photostability, extinction
coefficients and quantum yields, tunable excitation and emis-
sion spectra)1 and facile conversion into probes for positron
emission tomography (PET) imaging.2 However, one important
shortcoming of BODIPY dyes is their relatively small Stokes
shis, which can lead to experimental limitations, such as self-
quenching3 or direct acceptor excitation in FRET probes.4
Herein we report the rst systematic generation of MegaStokes
BODIPY dyes. We prepared a combinatorial library by derivati-
zation of a bifunctional BODIPY scaffold using copper-catalyzed
azide–alkyne cycloaddition (CuAAC) and nucleophilic substi-
tution. The incorporation of different triazole moieties paired
with a piperidinyl substituent at positions 3 and 5 of the
BODIPY core (Scheme 1) afforded 40 BODIPY-triazoles (so-
called BoDipy Click, BDC) with very large Stokes shis (i.e. from
74 to 160 nm). Further modication of BDC compounds led to
following our previously reported solid-phase strategy, which
avoids tedious purication steps and renders BODIPY
compounds in high purities.5
Weber and co-workers pioneered the preparation of envi-
ronmentally sensitive dyes by modifying opposite ends of
aromatic systems with electron donor and acceptor groups.6
This approach has been successfully applied to the synthesis of
numerous environmentally sensitive uorophores (e.g. dansyl,
NBD, ANS, aminophenoxazone).7 We envisioned that BDC
compounds, which include an electron-donating piperidinyl
group and 40 wide-ranging triazoles at opposed ends of the
BODIPY core, may behave as environmentally sensitive
compounds and recover the inherent uorescence of the
BODIPY core in specic environments. From the examination of
our 120-member library, we identied BDC-9 as a highly envi-
ronment-sensitive dye, and demonstrated its potential as a
species-selective and FA1 site-specic human serum albumin
(HSA) sensor.
2 Results and discussion
aDepartment of Chemistry, National University of Singapore, 3 Science Drive 3,
117543, Singapore. E-mail: chmcyt@nus.edu.sg; Fax: +65 6779 1691; Tel: +65 6779 Design and synthesis
7794
CuAAC is highly favoured for combinatorial purposes due to its
high efficiency, robustness and mild reaction conditions.8 The
bGraduate School for Integrative Sciences and Engineering, National University of
Singapore, Centre for Life Sciences, Singapore
cMRC Centre for Inammation Research, Queen's Medical Research Institute,
resulting triazole ring is chemically stable and, when properly
University of Edinburgh, EH16 4TJ Edinburgh, UK. E-mail: mvendrel@staffmail.ed. located within a uorescent core, can modulate the spectral
wavelengths of the resulting uorescent derivatives.9 A survey of
the literature revealed that the incorporation of electron-
donating and withdrawing groups at positions 3 and 5 of the
BODIPY scaffold can effectively alter its Stokes shis.10 Based on
ac.uk; Fax: +44 (0)1312426578; Tel: +44 (0)1312426685
dLaboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium,
Agency for Science, Technology and Research (A*STAR), Singapore
† Electronic supplementary information (ESI) available: General procedures,
chemical structures and characterization data of BDC, BDCAC and BDCCA. Full
these reports, we designed a trifunctional BODIPY aniline (4,
Scheme 1) for the synthesis of our library. This scaffold
characterization data (NMR, HR-MS) for BDC-9. Additional experimental data
for BDC-9–HSA binding. See DOI: 10.1039/c3sc22166k
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alkynes were used as building blocks for the construction of the
library. Final cleavage with 0.5% triuoroacetic acid (TFA) in
CH2Cl2 rendered 40 BDC derivatives with average purities of
95% aer minimum purication steps. Subsequent acetylation
and chloroacetylation of the aniline group afforded the corre-
sponding BDCAC and BDCCA compounds (see Fig. S8 and
Tables S2–S4 in ESI† for chemical structures and detailed
characterization).
General photophysical properties of BODIPY-triazoles
Most BODIPY-triazoles exhibited similar absorbance wave-
length maxima (BDC: 465–478 nm; BDCAC and BDCCA: 458–
468 nm), whereas their emission maxima spanned across a
much broader range (BDC: 546–616 nm; BDCAC and BDCCA:
542–612 nm) (Tables S2–S4 in ESI†). Notably, we observed that
the spectral properties of MegaStokes BODIPY dyes might be
ne-tuned with different alkyne building blocks. For instance,
electron-rich alkynes (e.g. 4-ethynyl-N,N-dimethylaniline)
generated compounds with longer absorption wavelengths than
alkynes containing electron-withdrawing groups (e.g. 4-(tri-
uoromethoxy)phenylacetylene) (Fig. S8–S10 in ESI†). While
BDC compounds exhibited low quantum yields in polar solvents
due to photoinduced electron transfer (PeT) effect,14 the acety-
lation and chloroacetylation of the aniline group partially
recovered their uorescence emission with an average 6-fold
uorescence increase. These properties affirm the potential of
BDC compounds to behave as uorescent turn-on sensors.
Scheme 1 Synthesis of the BDC library. Reagents and conditions: (a) pyrrole,
TFA, rt, 3 h; (b) NCS, anhydrous THF, ꢀ78 ꢁC to rt, 3 h; (c) DDQ, dry CH2Cl2, rt, 1 h;
(d) BF3$OEt2, anhydrous CH2Cl2, rt, 2 h; (e) Fe activated by 1 M aqueous HCl,
CH3OH–CH3COOH (10 : 1), reflux, 15 min; (f) CTC-PS resin, DIEA, CH2Cl2–DMF, rt,
24 h; (g) NaN3, DMF, rt, 30 min; (h) DMF–piperidine (4 : 1), rt, 45 min; (i) R–C^CH,
CuI, ascorbic acid, rt, 30 min; (j) 0.5% TFA in CH2Cl2, 10 min; (k) R2COCl, sat.
NaHCO3, rt, 10 min.
Solvatochromic effects
BODIPY dyes substituted at positions 3 and 5 have been
reported to display absorption and emission shis correlated to
solvent polarity.16 In order to examine the potential of BODIPY-
triazoles as environmentally sensitive uorophores, we
analyzed the absorbance and uorescence spectra of a repre-
possesses two electrophilic sites at positions 3 and 5, which can sentative MegaStokes BODIPY-triazole (BDC-9, Fig. 1) in
be modied via CuAAC and nucleophilic substitution,11 and one different solvents covering a wide range of polarities and
aniline group to construct the library using solid-phase viscosities (Table 1).
synthesis. Amino-functionalized BODIPYs can be successfully
The shape of the absorption spectrum of BDC-9 is similar to
loaded onto chlorotrityl chloride polystyrene resin (CTC-PS), most BODIPY dyes. Two absorption bands are observed: a S0–S1
which allows their cleavage under weakly acidic conditions transition band with a maxima spanning across 465 to 515 nm
and preserve the integrity of the uorophore (Scheme 1).5 and a weaker S0–S2 band at approximately 400 nm (Fig. 2a).
The aniline group also provided chemical stability to the These bands are less distinct in polar solvents due to band
BODIPY core1e and an additional point for further chemical broadening by solvent interactions. The absorption band cor-
derivatization.
responding to the S0–S1 transition showed a pronounced
The synthesis of 4 consisted of four steps with an overall dependence on the solvent polarity, and absorption maxima
yield of 37%. 1 was readily prepared from p-nitrobenzaldehyde shied to shorter wavelengths from hexane (515 nm) to aceto-
and pyrrole following reported methods.12 Chlorination with N- nitrile (465 nm) (Fig. 2a, Table 1). This shi suggests that the
chlorosuccinimide (NCS) followed by oxidation with DDQ dipole moment of the ground state may be larger than in the
afforded 2, which was subsequently treated with BF3$OEt2 to excited state. We detected a similar phenomenon in mixtures of
obtain the highly stable BODIPY analogue 3.12b,13 Reduction cyclohexane and ethanol (see Fig. S1a in ESI†). In very polar
with activated iron rendered 4 in quantitative yields. The aniline solvents such as DMSO and water, a slight reversal to longer
was then loaded onto CTC-PS resin using standard coupling wavelengths was detected. On the other hand, the position of
conditions. Treatment with sodium azide followed by piperi- the S0–S2 transition band was less affected by the solvent
dine generated a monosubstituted intermediate upon which polarity. These environment dependence shis in the absorp-
diversity was introduced via CuAAC. 40 chemically diverse tion maximum may be attributed to strong dipolar interactions
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BDC-9 in cyclohexane–ethanol mixtures, and observed that the
emission intensity decreased as solvent polarity increased from
cyclohexane to ethanol (see Fig. S1b in ESI†). Secondly, we
measured the emission spectra of BDC-9 in saturated alcohols
with different viscosity, and noticed a strong correlation
between uorescence intensity and viscosity (see Fig. S2 in
ESI†). Increased solvent viscosity reduces bond rotation at
positions 3 and 5 of the BODIPY core, which might minimize
non-radiative energy loss and lead to higher quantum yields.11e
The strong environment dependence of BDC compounds
conrms their potential as environmentally sensitive turn-on
sensors.
Sensor development
Next we examined the uorescence properties of BDC, BDCAC
and BDCCA to identify environmentally sensitive compounds
that could restore the strong uorescence of BODIPY in specic
environments. We performed a primary screen of our library
(Table S1 in ESI†), and observed that a number of BODIPY-tri-
azoles showed a signicant turn-on effect upon interaction with
the hydrophobic regions of different proteins. From this
screening we selected the compound BDC-9, which displayed a
remarkable selectivity towards HSA when compared to other
proteins and analytes (Fig. 3).
Fig. 1 Normalized absorbance and emission spectra of a representative BOD-
IPY-triazole (BDC-9) in DMSO at rt. The corresponding spectra of a reported
BODIPY (in DMSO) (BD-27) are also included for comparison.15 Blue: BD-27;
Orange: BDC-9.
In addition to its high selectivity, BDC-9 showed a signicant
220-fold increase in uorescence upon binding to HSA (Fig. 4),
and a hypsochromic shi of its emission maximum wavelength
(from 585 to 575 nm) (see Fig. S4 in ESI†). The response of BDC-
9 proved to be linear within a dynamic range between 0.37 and
31 mg mLꢀ1, and we determined the limit of detection of HSA
(S/N ¼ 3) as 0.3 mg mLꢀ1 (see Fig. S5 in ESI†).
from the asymmetric and charged-separated resonance forms of
the BDC structure (see Fig. S3 in ESI†).
We further investigated the selectivity of BDC-9 towards
serum albumins from different species. Serum albumins
represent a family of proteins with a high degree of similarity in
their primary sequence across various species (ꢂ75%
homology).18 However, some albumin ligands have been found
to show species-dependent binding differences, and the devel-
opment of species-selective probes may be useful for structural
The uorescence maximum wavelength of BDC-9 displayed
minor changes in different organic solvents (Table 1), whereas
its quantum yield (FF) showed a remarkable dependence on
the nature of the solvent (Fig. 2b, Table 1). In order to examine
the contribution of solvent polarity and viscosity in this
dependency, we rst analysed the uorescence intensity of
Table 1 Photophysical properties of BDC-9 in various solventsa
Dielectric
constant, 3
Viscosity,
h/mPa s
3max/
Solvent
labs,max/nm
104 Mꢀ1 cmꢀ1
lem,max/nm
FF (%)
D~n/cmꢀ1
Hex
CyH
Tol
CHCl3
EA
1.88
2.02
2.38
4.81
6.02
37.5
46.7
3.4
13.3
18.0
24.6
33.0
80.1
0.29
0.90
0.55
0.54
0.43
0.34
2.00
7.6617
4.5917
2.59
1.08
0.54
0.89
515
515
495
485
483
465
472
490
485
480
475
465
480
1.7
1.5
2.5
2.5
2.5
2.6
2.5
2.5
2.5
2.6
2.6
2.4
1.5
575
580
584
585
585
585
590
585
585
585
585
585
615
5.4
1.4
1.2
1.3
0.77
0.22
0.22
2.7
1.9
1.2
2027
2177
3079
3525
3610
4412
4148
3315
3525
3740
3959
4412
4574
ACN
DMSO
n-OctOH
n-HexOH
n-BuOH
EtOH
0.55
0.31
0.40
MeOH
H2O–MeOH (99 : 1)
a
Hexane: Hex; cyclohexane: CyH; toluene: Tol; ethyl acetate: EA; acetonitrile: ACN.
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marked selectivity only towards HSA with marginal response on
the other species.20
Analysis of the binding of BDC-9 at HSA
Following experiments were aimed at studying the binding
properties of BDC-9 on HSA. HSA contains two main drug
binding sites, namely Sudlow sites I and II.21 Recently, X-ray
crystallographic structures of HSA revealed at least three addi-
tional sites with affinity for drug compounds.22 These drug
binding sites correspond to the fatty acid (FA) sites 1, 3/4
(Sudlow site II), 5, 6 and 7 (Sudlow site I).23 In order to corrob-
orate the specic binding of BDC-9 and determine its binding
site, we performed competition assays using drugs binding to
the different sites (e.g. hemin (FA1 site),22b,k dansyl-L-norvaline
(FA3/4 sites),22i propofol (FA3/4 and FA5 sites),22g ibuprofen
(FA3/4 and FA6 sites)22d and warfarin (FA7 site)22d) were
performed.22b,24
As illustrated in Fig. 6a, only the competition with hemin
induced a signicant decrease in the uorescent response of
BDC-9 while the competition with all other drugs did not affect
its uorescence. In addition, the competition with hemin
reversed the hypsochromic shi observed upon HSA binding,
which conrms that hemin is able to displace BDC-9 from the
FA1 site (Fig. 6b). These observations clearly indicate that the
environmental turn-on response of BDC-9 is due to its binding
to the FA1 site (heme site) of HSA. There have been several
reports of uorescent dyes binding to FA3/4,25 FA626 and FA727
sites. However, to the best of our knowledge, this is the rst
experimentally proven uorescent dye binding to the FA1 site,28
and in which binding is not dependent on contacts with bound
lipid.25a BDC-9 is therefore a valuable probe for examining the
heme region on HSA.
Fig. 2 Spectra of a representative BODIPY-triazole (BDC-9, 100 mM) in various
organic solvents ordered according to polarity. Measurements were taken at rt. (a)
Normalized absorption spectra in various organic solvents; (b) emission spectra.
In addition, we performed a Job plot analysis to determine
the stoichiometry of the complex formed by BDC-9 and HSA,
and conrmed the results from site-selectivity studies. The
uorescence response of BDC-9 peaked at a 1 : 1 proportion of
BDC-9 : HSA, which indicates that BDC-9 binds mainly at one
site of the protein (see Fig. S6 in ESI†).29 A one-site binding
model was tted to the titration of HSA (0.67 mg mLꢀ1) with
serial concentrations of BDC-9, and we determined the disso-
ciation constant (KD) of BDC-9 as 12.7 ꢃ 0.4 mM (see Fig. S7 in
ESI†).
Application of BDC-9 to quantify HSA in urine samples
In order to examine the behaviour of BDC-9 as an HSA sensor in
complex matrices, we evaluated the application of BDC-9 to
quantify the amount of HSA in urine samples. The detection
and quantication of HSA in biouids is of great clinical
Fig. 3 Fluorescence response (F) of BDC-9 (10 mM) toward 16 different proteins
(0.13, 0.25, 0.50 and 1.00 mg mLꢀ1); 10 mM HEPES buffer (pH ¼ 7.4) (lexc: 460
nm, lem: 575 nm). F0 is the fluorescence intensity of BDC-9 in HEPES buffer. Values
are represented as means (n ¼ 4).
importance.30 Microalbuminuria, which involves an albumin
excretion rate of 15–40 mg mLꢀ1
, is a well-established cardio-
31
vascular risk marker and an indication of liver and renal
studies of albumin binding sites.19 We evaluated the uores- disease.32 We analysed the uorescence response of BDC-9 in
cence response of BDC-9 against serial concentrations of serum urine that was spiked with different amounts of HSA, up to 1 mg
albumins from rat (RSA), porcine (PSA), rabbit (RbSA), sheep mLꢀ1. We observed an excellent linear correlation between the
(SSA) and bovine (BSA). As shown in Fig. 5, BDC-9 displayed a uorescence response of BDC-9 and the amount of HSA in a
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shis, and represents the rst systematic generation of Mega-
Stokes dyes based on the BODIPY scaffold. The uorescence
emission of BDC compounds shows a strong dependence on
environmental properties, such as solvent polarity and viscosity.
We screened our library and identied BDC-9 as a highly
sensitive and species-selective HSA chemosensor. BDC-9
showed a remarkable 220-fold uorescence increase, species-
selectivity over other serum albumins and binding site speci-
city at the FA1 site (heme site) of HSA. Furthermore, we proved
that BDC-9 can be used for clinical applications to quantify HSA
in urine samples within the concentration limits of micro-
albuminuria. Altogether, these results validate MegaStokes
BODIPY dyes as new uorophores for the development of
environmentally sensitive uorescent probes.
Fig. 4 Fluorescence response of BDC-9 (10 mM) upon incubation with serial
dilutions of HSA (from 1.2 ꢄ 10ꢀ4 to 4 mg mLꢀ1) in 10 mM phosphate buffer
(pH ¼ 7.3); lexc: 460 nm. FF (in 4 mg mLꢀ1 HSA) ¼ 0.41. Values are represented as
means (n ¼ 3). Measurements were taken at rt.
4 Experimental
Materials
All commercially available reagents, solvents and proteins were
purchased from Sigma Aldrich, Alfa Aesar, Fluka, Merck or
Acros, and used as received unless otherwise stated. CH2Cl2
(Fisher Scientic, analytical grade) was freshly distilled from
P2O5 under nitrogen. Anhydrous THF was purchased from Alfa
Aesar and used without further purication. Phosphate buffer
(10 mM) was prepared following a modied recipe of Dulbecco's
PBS buffer (1ꢄ): 0.2 g KH2PO4, 2.17 g Na2HPO4$7H2O, 1000 mL
MilliQꢀ H2O, pH 7.3.
Measurements and analysis
Spectroscopic and quantum yield data were measured on a
SpectraMax M2 spectrophotometer (Molecular Devices). Data
analysis was performed using GraphPrism 5.0. Mixtures of BDC-
9 with HSA were prepared in phosphate buffer (pH 7.3, 1%
DMSO) at room temperature and uorescence measurements
were taken aer incubation for 2 h unless otherwise stated.
Analytical characterization was performed on a HPLC-MS (Agi-
lent-1200 series) with a DAD detector and a single quadrupole
mass spectrometer (6130 series) with an ESI probe. Analytical
HPLC method: eluents, A: H2O (0.1% HCOOH), B: CH3CN (0.1%
HCOOH), gradient 5% B to 95% B (10 min). Reverse-phase
Phenomenex C18 Luna column (4.6 ꢄ 50 mm2, 3.5 mm particle
Fig. 5 Species selectivity of BDC-9. (a) Fluorescence response of BDC-9 (10 mM)
upon interaction with serial dilutions of the respective albumins (from 1.2 ꢄ 10ꢀ4
to 4 mg mLꢀ1) in 10 mM phosphate buffer (pH ¼ 7.3); lexc: 460 nm, lem: 575 nm.
Values are represented as means and error bars as standard deviations (n ¼ 3).
Measurements were taken at rt. (b) Photographic image of BDC-9 (10 mM) mixed
with respective albumins. Irradiation with a handheld UV lamp at 365 nm.
1
size), ow rate: 1 mL minꢀ1. H-NMR, 19F-NMR and 13C-NMR
spectra were recorded on Bruker ACF300 (300 MHz) and
1
AMX500 (500 MHz) spectrometers and reported in d (ppm). H
NMR spectra were referenced to residual proton signals in
CDCl3 (d ¼ 7.26); 13C NMR spectra were referenced to solvent
resonances of CDCl3 (d ¼ 77.0 ppm); 19F NMR spectra were
referenced to external TFA (d ¼ ꢀ76.55 ppm vs. CFCl3 at 0.00
ppm).
clinically signicant range (Fig. 7), which proves the potential of
BDC-9 to quantify HSA levels in urine samples.
Quantum yield measurements. Quantum yields were calcu-
lated by measuring the integrated emission area of the uo-
3 Conclusions
In conclusion, we prepared a solid-phase 120-member library of rescent spectra in its respective solvents and comparing to the
BODIPY-triazoles (BDC, BDCAC and BDCCA) via CuAAC with area measured for Acridine Yellow (FF ¼ 0.47) in EtOH (h ¼
minimum purication steps and high purities. The incorpora- 1.361) when excited at 430 nm. Quantum yields for the BDC,
tion of triazole and piperidinyl moieties at positions 3 and 5 of a BDCAC and BDCCA libraries were calculated using the
bifunctional BODIPY core resulted in dyes with very large Stokes equation:
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ꢀ
ꢁꢀ
ꢁꢀ
ꢁ
Fsample
Freference
hsample
hreference
Absreference
Abssample
FsFample ¼ FFreference
where F represents the area of uorescent emission, h is the
refractive index of the solvent, and Abs is absorbance at the
excitation wavelength selected for standards and samples.
Emission was integrated between 460 and 750 nm.
In vitro uorescence screening. BODIPY-triazoles (10 mM)
were screened in 10 mM HEPES buffer, pH 7.4, containing 1%
DMSO. Fluorescence intensities were measured using a Spec-
traMax M2 plate reader in 384-well plates. Excitation was
provided at each compound's excitation range, and emission
spectrum was obtained starting from at least 30 nm beyond the
excitation wavelength. All the analytes were tested at four serial
concentrations (see Table S1 in ESI†).
Determination of dissociation constant for BDC-9 with HSA.
HSA (10 mM) was titrated against serial concentrations of BDC-9
(0.47 to 60 mM) and the uorescence intensities were measured
at 575 nm (lexc ¼ 460 nm). The uorescence intensity of bound
BDC-9 at each concentration was determined using the
equation:
FsatðF ꢀ F0Þ
Fbound
¼
ðFsat ꢀ F0Þ
where F and F0 are the uorescence intensities of a given
concentration of BDC-9 with and without HSA respectively. Fsat
is the uorescence intensity at the same concentration of BDC-9
when fully bound.
Fig. 6 Competition with site-selective HSA-binding drugs. HSA (fatty-acid free)
(0.34 mg mLꢀ1, 5 mM) was preincubated at rt for 2 h in 10 mM phosphate buffer
(pH ¼ 7.3) with different concentrations of the drugs (up to 200 mM) before BDC-
9 (10 mM) was added. (a) Fluorescence fold-change at 575 nm. F is the fluores-
cence intensity at the indicated drug concentration and F0 is the fluorescence
intensity with no drug added. (b) Red-shift in emission spectra of BDC-9 upon
competition with hemin (FA1 site); lexc: 460 nm. Values are represented as means
and error bars as standard deviations (n ¼ 3). Measurements were taken at rt.
Synthesis
General procedure for the preparation of the BDC library. 2-
Chlorotrityl chloride resin (220 mg, 0.275 mmol) was swollen in
CH2Cl2 (3.0 mL) for 15 min. A solution of 4 (30 mg, 0.085 mmol)
and DIEA (200 mL, 1.15 mmol) in DMF–CH2Cl2 (1 : 1, 2 mL) was
added to the resin suspension and shaken at rt for 24 h, aer
which the resin was capped with MeOH (0.4 mL, 1.8 mL gꢀ1
resin) and DIEA (100 mL) for 12 h. The resin was ltered, washed
with DMF (4 ꢄ 10 mL) and CH2Cl2 (4 ꢄ 10 mL) and dried. The
loaded resin was resuspended in CH2Cl2 and shaken at rt for
15 min and washed with DMF. A suspension of NaN3 (50 mg,
0.77 mmol) in DMF (3.0 mL) was added and the reaction
mixture further shaken at rt for 30 min. Aer washing with DMF
(4 ꢄ 10 mL), a solution of DMF–piperidine (4 : 1) (4.0 mL) was
added and the reaction shaken for 45 min. The alkyne (0.854
mmol), CuI (65 mg, 0.34 mmol) and ascorbic acid (60 mg, 34
mmol) were subsequently added and the reaction mixture
shaken for another 30 min. The resin was ltered, washed with
DMF (4 ꢄ 10 mL) and CH2Cl2 (4 ꢄ 10 mL) following which
cleavage was performed with 0.5% TFA in CH2Cl2 (3 ꢄ 15 mL,
10 min each). The organic extracts were recovered and
concentrated in vacuo. The residue was puried by ash column
chromatography on silica gel (eluent: CH2Cl2 to CH2Cl2–MeOH
(98 : 2)) to afford the corresponding BDC library compound as
an orange solid (ca. 16 mg, 0.03 mmol, 70% yield) (see Table S2
in ESI† for chemical structures and detailed characterization).
General procedure for the preparation of BDCAC and
Fig. 7 Fluorescence response of BDC-9 in urine samples. HSA (0–1 mg mLꢀ1
)
was added to 10% urine from healthy individuals in 10 mM phosphate buffer
(pH ¼ 7.3). BDC-9 was added at 10 mM concentration; lexc: 460 nm, lem: 575 nm.
Measurements were taken at rt. Values represented as means (n ¼ 3) and error
bars as standard deviations; linear regression: R2 ¼ 0.998.
BDCCA libraries. To
a solution of the respective BDC
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3,5-Dichloro-8-(40-aminophenyl)-4,4-diuoro-4-bora-3a,4a-
diaza-s-indacence (4). A suspension of iron powder (1.46 g, 26.2
mmol) was activated in 1 M aqueous HCl for 1 min, rinsed with
absolute EtOH and used as such. A solution of 3 (500 mg, 1.3
mmol) in EtOH (80 mL) and acetic acid (8 mL) was added the
activated iron and reuxed. The reaction mixture was moni-
tored by TLC. Upon completion, iron was removed and the
solvent was evaporated in vacuo. The residue was diluted with
water and extracted with CH2Cl2 (3 ꢄ 70 mL). The combined
organic extracts were washed with saturated Na2CO3, saturated
brine, dried over anhydrous Na2SO4 and the solvents removed
in vacuo. The residue was puried by ash column chroma-
tography on silica gel (hexane–EtOAc–MeOH–NH3: 6 : 3 : 1 :
0.05) to afford 4 as a dark red solid (197 mg, 0.56 mmol, 98%
yield); mp 134–135 ꢁC. 1H NMR (300 MHz, CDCl3): d 7.34 (dt, J ¼
8.5, 2.6 Hz, 2H), 6.93 (d, J ¼ 3.8 Hz, 2H), 6.76 (dt, J ¼ 8.5, 2.6 Hz,
2H), 6.43 (d, J ¼ 3.8 Hz, 2H), 4.08 (br s, 2H); 13C NMR (75 MHz,
CDCl3): d 149.7, 144.9, 143.2, 133.4, 132.7, 131.1, 122.3, 118.1,
114.4; 19F NMR (282 MHz, CDCl3): d ꢀ148.65 (q, J ¼ 28 Hz);
HRMS (C15H11BCl2F2N3): calc. [M + H]+: 352.0386, found [M +
H]+: 352.0215.
10-(4-Aminophenyl)-5,5-diuoro-7-(4-phenyl-1H-1,2,3-triazol-
1-yl)-3-(piperidin-1-yl)-5H-dipyrrolo[1,2-c:20,10-f][1,3,2]diazabor-
inin-4-ium-5-uide (BDC-9). Orange solid (17 mg, 0.032 mmol,
72% yield); mp 129–131 ꢁC. 1H NMR (500 MHz, CDCl3): d 8.56 (s,
1H), 7.95 (d, J ¼ 6.9 Hz, 2H), 7.45 (t, J ¼ 7.6, Hz, 2H), 7.34 (t, J ¼
7.6 Hz, 1H), 7.27 (d, 8.2 Hz, 2H), 6.94 (d, J ¼ 5.7 Hz, 1H), 6.80 (d,
J ¼ 8.2 Hz, 2H), 6.62 (d, J ¼ 3.8 Hz, 1H), 6.42 (d, J ¼ 3.8 Hz, 1H),
6.32 (d, J ¼ 5.1 Hz, 1H), 5.30 (s, 1H), 3.78–3.88 (m, 4H), 1.64–1.80
(m, 6H); 13C NMR (75 MHz, CDCl3): d 162.2, 146.7, 136.0, 135.7,
134.9, 131.8, 131.3, 131.0, 130.8, 129.6, 128.7, 127.9, 126.0,
125.0, 122.4, 122.3, 116.9, 115.4, 115.0, 114.9, 109.7, 53.4, 52.0,
26.3, 24.0; 19F NMR (282 MHz, CDCl3): d ꢀ132.37 (q, J ¼ 35 Hz);
HRMS (C28H27BF2N7): calc. [M + H]+: 510.2384, found [M + H]+:
510.2399.
compounds (6 mg, ca. 0.011 mmol) in CH2Cl2 (1.5 mL), 6 drops
of saturated aqueous NaHCO3 were added and cooled to 0 C.
ꢁ
Acetyl chloride (10 mL, 0.11 mmol) was added in portions over 1
min and the resulting mixture was stirred at rt and monitored
by analytical TLC. Upon reaction completion, the reaction was
diluted with CH2Cl2 (15 mL), washed with water (2 ꢄ 15 mL),
saturated NaHCO3 (1 ꢄ 15 mL), saturated brine (1 ꢄ 15 mL) and
dried over anhydrous Na2SO4. The organic extract was evapo-
rated to afford the corresponding BDCAC or BDCCA
compounds as an orange solid (ca. 6 mg, >90% yield) (see
Tables S3 and S4 in ESI† for chemical structures and detailed
characterization).
2,20-((4-Nitrophenyl)methylene)bis(1H-pyrrole) (1). To
a
solution of 4-nitrobenzaldehyde (1.0 g, 6.6 mmol) in pyrrole
(10 mL, 144 mmol) was added TFA (0.51 mL, 0.66 mmol) and
stirred at rt for 3 h. The mixture was concentrated in vacuo, and
puried by ash column chromatography on silica gel (hexane–
EtOAc, 4 : 1) to afford 1 as a bright yellow solid (1.63 g, 6.10
mmol, 92% yield). 1H NMR (300 MHz, CDCl3): d 8.17 (d, J ¼ 8.7
Hz, 2H), 8.00 (br s, 2H), 7.38 (d, J ¼ 8.7 Hz, 2H), 6.75 (d, J ¼
5.8 Hz, 2H), 6.18 (dd, J ¼ 5.8, 2.9 Hz, 2H), 5.87 (br s, 2H), 5.58 (s,
1H); 13C NMR (75 MHz, CDCl3): d 150.4, 147.7, 131.5, 129.9,
124.5, 118.7, 109.5, 108.5, 44.5; MS (ESI): m/z [M + H]+ ¼ 268.1.
1,10-Dichloro-5-(4-nitrophenyl)dipyrromethene (2).13 A solu-
tion of 1 (1.1 g, 4.1 mmol) in anhydrous THF (35 mL) was stirred
under N2 atmosphere at ꢀ78 ꢁC for 15 min. N-Chlor-
osuccinimide (1.4 g, 10.3 mmol) in anhydrous THF (35 mL) was
added dropwise using a pressure equalizing funnel and the
resulting mixture was stirred at rt for 3 h. Upon completion, 2,3-
dichloro-5,6-dicyano-p-benzoquinone (1.12 g, 4.92 mmol) was
added and the reaction mixture stirred at rt for an additional
2 h. THF was removed in vacuo and the resulting mixture
was diluted with water (20 mL) and extracted with CH2Cl2 (3 ꢄ
70 mL). The combined organic extracts were washed with
saturated brine, dried over anhydrous Na2SO4 and the solvents
removed in vacuo. The resulting residue was puried by ash
column chromatography on silica gel (hexane–EtOAc, 10 : 1) to
afford 2 as a dark orange solid (0.78 g, 2.3 mmol, 57% yield). 1H
NMR (300 MHz, CDCl3): d 8.33 (d, J ¼ 4.3 Hz, 2H), 7.63 (d, J ¼ 4.3
Hz, 2H), 6.42 (d, J ¼ 2.1 Hz, 2H), 6.29 (d, J ¼ 2.1 Hz, 2H); 13C
NMR (75 MHz, CDCl3): d 149.1, 143.7, 142.7, 138.7, 137.1, 132.3,
130.1, 123.8, 118.5; MS (ESI): m/z [M + H]+ ¼ 334.0.
Acknowledgements
The authors acknowledge the nancial support from the
intramural funding by A*STAR Biomedical Research Council, a
Singapore Ministry of Education Academic Research Fund Tier
2 (MOE2010-T2-1-025) and the receipt of a NGS Scholarship (E.
J. C). M. V. acknowledges the support by the Medical Research
Council. The authors also thank Dr Zhang Liyun (Hefei Insti-
tutes of Physical Science, Chinese Academy of Sciences),
Roopsha Brahma and Prof. Chu-Young Kim (National Univer-
sity of Singapore) for their great help and advice.
3,5-Dichloro-8-(40-nitrophenyl)-4,4-diuoro-4-bora-3a,4a-
diaza-s-indacence (3).13 To a solution of 2 (1.0 g, 2.97 mmol) in
anhydrous CH2Cl2 (70 mL) was added N,N-diisopropylethyl-
amine (3.1 mL, 17.8 mmol) stirred at 0 ꢁC for 10 min. Boron
triuoride diethyl etherate (2.2 mL, 17.8 mmol) was added
dropwise and the resulting mixture was stirred at rt for 2 h.
Upon completion, solvents were removed in vacuo and the
residue puried by ash column chromatography on silica gel
(hexane–EtOAc–MeOH, 10 : 1 : 0.1) to afford 3 as a deep
Notes and references
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