Copper(II)-induced Fluorescence Quenching of a BODIPY Fluorophore

Article abstract of DOI:10.1002/zaac.201800154

The ongoing developments in fluorescence microscopy have pushed the interest in new fluorescent probes with specific properties. Aside of controlling emissive states by light irradiation, defined control by changing the chemical environment has come into focus. In this context, we designed a fluorescent probe by conjugation of bipyridine, acting as sensor for Cu^II, to a BODIPY derivative at the shortest possible distance between sensor and chromophore. Herein, we present the synthesis of the BODIPY probe along with its crystal structure. We found a strong dependence of absorption and emission upon Cu^II complex formation decreasing the fluorescence quantum yield to ca. 1 % in respect to the pure probe. We hypothesize that further functionalization with a linker could make this compound and interesting probe for fluorescence microscopy.

Full text of DOI:10.1002/zaac.201800154

Title: CuII-induced fluorescence quenching of a BODIPY fluorophore
Authors: Dirk-Peter Herten, Andreas Haderspeck, Felix Braun, and
Hubert Wadepohl
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To be cited as: Z. anorg. allg. Chem. 10.1002/zaac.201800154
Link to VoR: http://dx.doi.org/10.1002/zaac.201800154

10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
CuII-induced fluorescence quenching of a BODIPY fluorophore
Dirk-Peter Herten*[a], Andreas Haderspeck[a], Felix Braun[a], Hubert Wadepohl[b]
Dedicated to Professor Peter Comba on the Occasion of his 65th Birthday.
Abstract: The ongoing developments in fluorescence microscopy
have pushed the interest in new fluorescent probes with specific
properties. Aside of controlling emissive states by light irradiation,
defined control by changing the chemical environment has come into
focus. In this context, we designed a fluorescent probe by conjugation
of bipyridine, acting as sensor for CuII, to a BODIPY derivative at the
shortest possible distance between sensor and chromophore. Here,
we present the synthesis of the BODIPY-probe along with its crystal
structure. We found a strong dependence of absorption and emission
upon CuII-complex formation decreasing the fluorescence quantum
yield to ca. 1% in respect to the pure probe. We hypothesize that
further functionalization with a linker could make this compound and
interesting probe for fluorescence microscopy.
To become effective, fluorescent probes should emit as much
photons as possible upon excitation which points to the
importance of choosing the fluorescent dye. Here, the important
parameters are molecular brightness and photo-stability where
the molecular brightness is the product of extinction coefficient
and quantum yield. Good fluorophores usually show extinction
coefficients of more than 100,000 M-1cm-1 and a quantum yield of
90% or higher. It is commonly known that high quantum yields are
achieved when non-radiative transitions e.g. by internal
conversion (IC) or intersystem crossing (ISC) are suppressed.
Rigid structures lacking flexibility, like conjugated aromatic rings,
or substituents, like Fluorine, are therefore favoured for the design
of fluorescent probes, while substituents, like Bromine or Iodine,
lead to a significant reduction of the quantum yield due to the
heavy atom effect.[19] Photo-stability is governed by chemical
reactions occurring in the excited state. While many of these
reactions may occur from the triplet state, i.e. by spin exchange
and subsequent reaction with triplet oxygen, it is also known that
excitation at short wavelength (UV) can lead to photo-induced
decomposition of fluorophores.
Beyond this, also a high Stokes shift and a narrow emission band
is of advantage as it simplifies optical separation of the emitted
light from scattered excitation. For designing fluorescent probes,
the sensor unit switching the emission, is of equal importance as
the fluorophore. When driven by metal ion coordination, not only
the quenching mechanisms play an important role but also the
kinetics of metal ion coordination. Additional requirements are
imposed by the envisioned application, e.g. for single-molecule
based super-resolution microscopy only a small fraction of
molecules must be in the on-state such that non-overlapping
point-spread functions can be imaged in each frame. The
achievable labelling density is also limited by residual
fluorescence emission of molecules in their off-state. Both effects
are directly governed by the properties of the ligand, i.e. its
switching kinetics and the quenching mechanism controlling the
fluorophore emission. For instance, in single-molecule
localization microscopy based on reversible metal ion
complexation the dissociation reaction plays an important role
because the on-state duration must match the integration times of
the image detector to maximise localisation precision whilst
minimizing the total image acquisition time to achieve a full
reconstruction. In this sense, Boron-dipyrromethene (BODIPY)
dyes have high extinction coefficients with relatively narrow
Introduction
In recent years, the interest in novel fluorescent probes increased
due to improved labelling approaches for cell experiments,[1] and
promising applications in microscopy.[2,3] What makes them
interesting is that the chemical environment can be used to gain
control of the spectroscopic states.
Fluorescent probes can be realized as turn-off or turn-on probes
reacting with an emission decrease or increase, respectively.
Aside of collisional quenching,[4] fluorescence can be controlled
by a variety of different mechanisms, like Förster resonance
energy transfer (FRET),[5] photo-induced electron transfer
(PET),[3,6] (twisted) internal charge transfer ((T)ICT),[7] or through
bond energy transfer (TBET).[8] The field gained interest as
[9]
isomerisation, long-lived dark states,[10] or adduct formation[11]
supported the advancements in super-resolution fluorescence
microscopy. Also, chemical transformations leading to changed
fluorescence emission have been widely studied, e.g.
intramolecular cyclization of rhodamine derivatives,[12] DNA
hybridization,[13] protonation,[14] imine tautomerization in photo-
acids,[15] reversible redox reactions,[16] enzymatic cleavage,[17]
and several more.[18]
[a]
[b]
Prof. Dr. Dirk-Peter Herten, Dr. Andreas Haderspeck, Felix Braun
Institute of Physical Chemistry
Heidelberg University
Im Neuenheimer Feld 229, D-69120 Heidelberg, Germany
E-mail: dirk-peter.herten@urz.uni-hd.de
Prof. Dr. Hubert Wadepohl
absorption bands in the visible range and are thereby interesting
[20]
candidates for metal ion sensors.
Due to their high quantum
yields and good photo-stabilities they are even used in single-
molecule fluorescence microscopy experiments. [21]
((INSERT SCHEME 1 HERE))
In previous studies, the bidentate bipyridine was found a suitable
ligand for designed CuII-dependent switchable fluorescent
probes.[2,22–25] Therefore, we designed a bipyridine functionalized
turn-off probe with the shortest possible distance to the
Institute of Inorganic Chemistry
Heidelberg University
Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany
This article is protected by copyright. All rights reserved.

10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
chromophore to maximize quenching upon coordination of CuII.
We synthesized a bipyridine/BODIPY conjugate BODIPY-BPY 1
(Scheme 1) and characterized its spectroscopic properties in
presence of CuII.
In contrast to the rather weak changes in absorbance, strong
quenching of fluorescence emission can be observed upon CuII
coordination (fig. 3). Quenching of the fluorescence happens
instantaneously upon addition of a CuSO4 accompanied by a
bathochromic shift of ca. 10 nm. Fluorescence emission is fully
recovered along with absorption upon addition of an excess of
EDTA supporting reversible coordination of CuII. Figure 4 shows
a Stern-Volmer plot of the titration with CuII. Here, F0/F is plotted
over the CuII concentration c(CuII), where F0 is the fluorescence
intensity of a pure BODIPY-BPY 1 solution and F is the
fluorescence intensity measured at a given c(CuII). The data
shows a sigmoidal shape levelling off close to F0/F = 94.4
reflecting a residual fluorescence emission of the CuII complex
with BODIPY-BPY 1 of ca. 1%. Similar observations have been
made when using methanol as solvent. Obviously, simple
complex formation cannot account for the observed sigmoidal
shape. One might speculate that two BODIPY-BPY 1 could be
coordinated to a single metal cation at low CuII concentrations but
this effect is not reflected in the absorbance data. More extensive
spectroscopic studies might shed light on this issue.
Results and Discussion
Synthesis and molecular structure
The well-known synthesis of symmetric BODIPY dyes starts with
condensation of substituted pyrroles with an aldehyde.[26–28] The
intermediate dipyrromethane is then oxidized, e.g. by a quinone,
and reacted with borontrifluoroetherate. Frequently, the pyrroles
are substituted with two alkyl groups in position 2 and 4 to
sterically prevent rotation of the residue introduced by the
aldehyde which otherwise leads to a reduced fluorescence
quantum yield. [27] Based on this route we synthesized a probe in
which the ligand bipyridine is directly coupled to the BODIPY
chromophore such that metal ion binding can influence their
spectroscopic properties.
((INSERT FIGURE 1 HERE))
Synthesis of BODIPY-BPY 1 starts with hydrolysis of 5-
(dibromomethyl)-2,2‘-bipyridine to form the corresponding
aldehyde, followed by BODIPY synthesis according to standard
procedures (Scheme 1). Purification and recrystallization yields
red crystals suited for crystallographic analysis (fig. 1). In
BODIPY-BPY 1 the bipyridine moiety is directly coupled to the
chromophore suggesting a strong influence on spectroscopic
states. On the other hand, the planes of bipyridine and
chromophore are orthogonally oriented due to steric hindrance by
the two methyl groups in 3 and 7 position preventing direct --
interaction. The nitrogen atoms in the two pyridine rings of the
bipyridine moiety are pointing in opposite directions and must be
reorganized upon coordination to metal ions, like CuII.
Spectroscopic characterization
The absorption spectrum of BODIPY-BPY 1 (fig. 2) shows the
BODIPY absorption band at 502 nm [29] along with aromatic side
bands (236 nm) and the typical bipyridine band at 285 nm.[26] In
methanol we found a molar extinction of 100,000 M-1cm-1 that is
reduced in MOPS buffered aqueous solution to ca.
79,000 M-1cm-1.
Upon addition of increasing CuII amounts absorbance is further
reduced by ca. 20% when reaching saturation also showing a
slight red shift by ca. 5 nm (fig. 2). At the same time the bipyridine
band at 285 nm shows a pronounced bathochromic shift by ca.
33 nm. Additionally, isosbestic points can be observed indicating
a second species, i.e. the CuII complex with the bipyridine moiety,
influencing the spectroscopic properties of BODIPY-BPY 1. CuII
coordination is reversible as the BODIPY absorbance (dashed
line) is fully recovered upon addition of excess EDTA.
((INSERT FIGURE 2 HERE))
Conclusions
Overall, we have synthesized and characterized
a new
fluorescent probe sensitive to CuII. The probe BODIPY-BPY 1
combines the bright emission of the known BODIPY fluorophore
with sensory activity of bipyridine. Since chromophore and
coordination site are in close proximity, we observed strong
spectroscopic effects by coordination of CuII on absorbance and
emission of the BODIPY moiety. We found that the quenching by
CuII is based on the coordination equilibrium and can be reverted
by addition of EDTA.
Although absorbance and emission are showing the same trend
upon addition of CuII, the strong quenching of fluorescence
emission cannot be justified by the change in absorbance alone.
It is well known that bipyridine itself doesn’t show fluorescence
emission because free rotation of the pyridine rings in solution
promote alternative decay mechanisms. In contrast to redox inert
metal cations, the CuII complex with bipyridine becomes not
fluorescent, although coordination by CuII stabilizes the two rings
in plane as indicated by the red shift of the bipyridine band.[30] This
observation points towards additional quenching effects by
interaction metal-ligand orbitals influencing the electron
distribution as well as mesomeric effects.
In previous studies of similar probes, we have observed
comparable effects and could exclude energy transfer as possible
quenching mechanism. We found that the overlap between dye
emission and ligand/complex absorption was too small to enable
energy transfer.[2,23–25,31] However, as complex and chromophoric
centre were always in close proximity, as is the case for BODIPY-
BPY 1, photo-induced electron transfer (PET) remains a possible
quenching mechanism. Further studies and an eventual proof of
PET in such systems could bring up interesting possibilities of
such probes for light-controlled redox states of CuII complexes,
e.g. in catalytic reactions.
((INSERT FIGURE 3 HERE))
BODIPY-BPY 1 itself is strongly fluorescent showing an emission
band at 517 nm typical for BODIPY derivatives. The relative
quantum yield in methanol was determined to be 24% using
fluorescein as standard.
((INSERT FIGURE 4 HERE))
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10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
122.28, 124.85, 131.11, 136.97, 137.25, 149.47, 151.69, 154.71, 160.61,
190.64. MS (ESI pos): m/z = 184.10 ([M+H]+).
Beyond this, functionalization of fluorescent metal indicators have
an interesting perspective in the field of microscopy, e.g. as
probes for super-resolutions microscopy or as additional
approach for multiplexing in microscopy to increase the number
X-Ray Crystal structure determination
of
simultaneously
observable
targets.[2,24]
Moreover,
Crystal data: BODIPY-BPY 1: C23H21BF2N4, Mr = 402.25, triclinic, space
group P-1, a = 9.3868(2), b = 9.7303(3), c = 10.8644(3) Å,  = 92.910(2),
 = 96.618(2),  = 103.217(3) °, V = 956.54(5) Å3, Z = 2, dcalc = 1.397 Mg·m-
3,  = 0.797 mm-1, F000 = 420. T = 120(1) K,  range 4.1 to 70.6 °. Index
ranges h, k, l ±11, ±11, ±13. Reflections measd.: 76817, indep.: 3580 [Rint
= 0.0456], obsvd. [I > 2(I)]: 3472. Final R indices [Fo > 4(Fo)]: R(F) =
0.0460, wR(F2) = 0.1195, GooF = 1.098. Difference density: max, min
0.267, -0.296 e·Å-3.
functionalized probes could be used for implementing sensory
layers in nanostructured materials adding function to certain
regions.
Experimental Section
Synthesis
Data collection: Agilent Technologies Supernova-E CCD diffractometer,
Cu-K radiation, microfocus X-Ray tube, multilayer mirror optics,
 = 1.5418 Å. Lorentz, polarization and numerical absorption correction
(max., min. transmission factors: 0.919, 0.862).[33] Structure solution: ab
initio charge flipping.[34] Refinement: full-matrix least squares methods
based on F2;[35] all non-hydrogen atoms anisotropic, most hydrogen atoms
located and refined (except those of the methyl groups, which were input
at calculated positions and refined as variable metric rigid groups).
General Methods. The BODIPY-BPY probe was synthesized starting with
alkaline hydrolysis of 5-(dibromomethyl)-2,2'-bipyridine to [2,2'-bipyridine]-
5-carbaldehyde as precursor in the well described BODIPY synthesis.[27,28]
Preparation of 9-(2,2’-bipyridine-5-yl)-1,3,5,7-tetramethyl boron
dipyrromethene (BODIPY-BPY) 1: 2,4-Dimethylpyrrol (97%, 1.38 g, 1.5
ml, 14.4 mmol) and (2,2'-bipyridine)-5-carbaldehyd 2 (1.34 g, 7.3 mmol)
are successively dissolved in 150 mL DCM (abs.) under inert-gas
atmosphere (Ar) at room temperature. After addition of catalytic amounts
of TFA the mixture is stirred for approx. 3 h. When the reaction is finished
(residual substrate checked by TLC), 2,3-Dichloro-5,6-dicyano-
pbenzochinon (98%, 1.65 g, 7.3 mmol in 50 ml DCM) is added and stirred
for another 10 min before addition of an excess of DIPEA (30.0 ml, 176.4
mmol) and BF3OEt2 (10.0 ml, 79.0 mmol). The reaction mixture is stirred
for another 2 h and then purified by extraction with water. After separation
of the organic phase it is dried over NaSO4 and the solvent is removed in
CCDC 1838079 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cambridge
Crystallographic
Data
Centre
via
https://www.ccdc.cam.ac.uk/data_request/cif.
1H- and 13C-NMR spectra: NMR spectra were recorded on a Varian
Mercury Plus 300 MHz spectrometer or Varian 500 MHz NMR
a
System. Peak shifts were reported relative to solvent peaks according to
Fulmer et al.[36] The chemical shift δ is stated in parts per million (ppm), the
coupling constants J in Hertz (Hz) in respect to 1H-1H-coupling. Signal
multiplicities are indicated by: s = Singulett, d = Dublett, dd = Dublett of
Dubletts, t = Triplett, q = Quartett, m = Multiplett.
a
rotary evaporator. The product is further purified by column
chromatography (SiO2; DCM/EtOAc 1:3) and recrystallization in
DCM/EtOAc (1:8) yielding red crystals. The yield was 24% based on (2).
C23H21BF2N4 (402.25581). Elemental analysis didn’t yield satisfying results
(C23H21BF2N4 (402.26); C 67.17 (calc. 68.68); H 5.53 (calc.5.26); N 13.04
(calc. 13.93) and has been reported being difficult for BODIPY derivatives
due to the presence of boronnitride.[32]. Therefore, purity of the compound
was proven by 1H-NMR and MS ESI (see supporting information) and low
amounts of the compound were further purified by HPLC for the
spectroscopic experiments. 1H-NMR (300 MHz, CDCl3) δ (ppm) = 8.72
(ddd, J=4.8, 1.9, 0.9, 1H, 2), 8.65 – 8.55 (m, 2H, 5, 12), 8.53 – 8.43 (m,
1H, 9), 7.93 – 7.82 (m, 1H, 6), 7.78 (dd, J=8.1, 2.3, 1H, 11), 7.37 (ddd,
J=7.6, 4.8, 1.2, 1H, 1), 6.02 (s, 2H, 20, 23), 2.57 (s, 6H, 28, 30), 1.46 (s,
6H, 27, 29). 13C-NMR (75 MHz, CDCl3) δ (ppm) = 156.76 (10), 156.45 (19,
24), 155.32 (4), 149.50 (2), 148.04 (12), 142.98 (14, 18), 137.67 (13),
137.27 (6), 137.20 (11), 131.65 (21, 22), 131.35 (7), 124.45 (1), 121.85 (9),
121.46 (5), 121.11 (20, 23), 77.58, 77.16, 76.74, 15.36 (27, 29), 14.80 (28,
30). MS (ESI pos): m/z = 403.30 ([M+H]+). UV/VIS (H2O): max (): 502
(0.53), 236 (0.28), 285 (0.18) nm.
Mass Spectrometry: Bruker ApexQe hybrid 9.4 T FT-ICR. For MALDI
spectra DCTB was used as matrix.
Optical Spectroscopy: All measurements were carried out in ultra-micro-
quarzglass cuvettes (105.251-QS, Hellma Analytics, Müllheim) with an
optical path length of 3mm and a minimum volume of 45 μl. UV/VIS
absorption was measured on
a “Cary 500 Scan” (Varian/Agilent
Technologies) against air. Spectra were corrected for absorption of the
cuvette and the pure solvent. Fluorescence spectra were taken on a “Cary
Eclipse 500” (Varian/Agilent Technologies) at an excitation wavelength
just below the absorption maximum to minimize light scattering.
Fluorescence spectroscopy was carried out at concentrations well below
10-3 M (usually at ca. 10-6 M) using 100 μl sample to avoid inner filter
effects.
Preparation of (2,2'-bipyridine)-5-carbaldehyde 2: A mixture of 5-
(dibromomethyl)-2,2‘-bipyridine (3 mg, 9.2 nmol) and CaCO3 (2.0 mg, 20.1
nmol) in 100 ml H2O is boiled for 6 h under reflux. After cooling to room
temperature the mixture is extracted three times with 100 ml DCM each.
The united organic phase is dried over NaSO4 before the solvent is
removed in a rotary evaporator. The product is further purified by column
chromatography (SiO2, PE/EtoAC 5:1). The yield was 85% based on (4).
C11H8N2O (184.19). 1H-NMR (600 MHz, CDCl3): δ (ppm) = 7 38 (t, 1J(H) =
12 Hz, 1 H), 7.86 (t, 1J(H) = 7 Hz, 1 H), 8.27 (d, 1J(H) = 8 Hz, 1 H), 8.49 (d,
1J(H) = 8 Hz, 1 H), 8.60 (d, 1J(H) = 8 Hz, 1 H), 8.71 (d, 1J(H) = 4 Hz, 1 H),
9.11 (s, 1 H), 10.15 (s, 1 H). 13C-NMR (150 MHz, CDCl3): δ (ppm) = 121.35,
Titrations: Titration with CuII was performed by stepwise addition of 1 µl
CuSO4 stock solution and careful mixing by loading the pipette multiple
times. Fluorescence spectra were recorded after equilibration.
Concentrations were corrected for the respective dilution in each titration
step. Fluorescence intensities were measured by integrating the emission
spectra over ± 10 nm around maximum emission to minimize noise
fluctuations.
Acknowledgements
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10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
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We grateful acknowledge funding by the Federal Ministry of
Education and Research (BMBF/VDI, Switch-Click). We thank
Prof. Markus Enders (Institute of Inorganic Chemistry, Heidelberg
University) for support with NMR spectroscopy and Prof. Jürgen
H. Gross for support with mass spectrometry. We also thank Prof.
Gregor Jung for helpful discussion.
Keywords: fluorescent switches • metal ion sensing •
fluorescence spectroscopy
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10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
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A fluorescent probe by conjugation
of bipyridine, acting as sensor for
CuII, to a BODIPY derivative at the
shortest possible distance between
Dirk-Peter Herten*, Andreas
Haderspeck, Felix Braun, Hubert
Wadepohl
+ EDTA
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sensor and chromophore.
CuII-induced fluorescence
quenching of a BODIPY fluorophore
+ CuII
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Author(s), Corresponding Author(s)*
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10.1002/zaac.201800154
Zeitschrift für anorganische und allgemeine Chemie
ARTICLE
Additional Author information for the electronic version of the article.
Dirk-Peter Herten: 0000-0002-3884-0262
Figure captions
Scheme 1. Syntheses of BODIPY-BPY 1.
Figure 1. Crystal structure of BODIPY-BPY 1.
Figure 2. Absorption spectra of BODIPY-BPY 1 (23 µM; yellow) in 10 mM MOPS buffered aqueous solution (max = 502 nm) and at increasing CuII concentrations
as indicated by colour shading from yellow to blue (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 14, 19 µM of CuSO4). While the BODIPY absorbance is only weakly shifted to 507 nm,
a strong bathochromic shift is visible for the bipyridine band from 285 to 317 nm. Addition of excess EDTA fully recovers the BODIPY absorption band (dashed line).
Figure 3. Emission spectra of BODIPY-BPY 1 (exc = 458 nm) in 10 mM MOPS buffered aqueous solution and at increasing CuII concentrations as indicated by
colour shading from yellow to blue (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 14, 19 µM of CuSO4). Excess of EDTA fully recovers the BODIPY emission (dashed line).
Figure 4. Stern-Volmer plot of the titration of BODIPY-BPY 1 (7 µM) in 10mM MOPS buffered aqueous solution with CuSO4. The titration yields a sigmoidal shape
levelling off at ca. 94.4 reflecting a residual fluorescence intensity of about 1% (dashed line shows the logistic function to guide the eye).
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