Pyrromethene dialkynyl borane complexes for "cascatelle" energy transfer and protein labeling

Article abstract of DOI:10.1002/anie.200500808

(Chemical Equation Presented) All aglow: A new class of highly luminescent dyes is built by the attachment of a pyrene fragment to the boron center of a boradiazaindacene moiety through an ethynyl unit (see scheme). Very efficient energy transfer from the pyrene to the indacene center produces virtual large Stokes shifts, which are maintained, along with strong luminescence (Φ = 32-45%) when the dyes are attached to proteins such as bovine serum albumin.

Full text of DOI:10.1002/anie.200500808

Communications
Fluorescence Imaging
Pyrromethene Dialkynyl Borane Complexes for
“Cascatelle” Energy Transfer and Protein
Labeling**
Gilles Ulrich,* Christine Goze, Massimo Guardigli,
Aldo Roda, and Raymond Ziessel*
The design of strongly luminescent probes is a thriving subject
which finds applications in electroluminescent devices and
fluorescence technology.[1,2] Fluorescent labels and probes
have found widespread uses in biomedicine, polymer science,
and sensor chemistry.[3,4] One of the most promising classes of
dyes is that of cyanines stabilized with BF2 fragments, such as
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy) deriva-
tives.[5] These non-ionic, stable molecules have exceptional
optical and luminescence properties,[6] and a wide range of
dyes have been engineered by modification of the pyrrole
substituents.[7] To date, chemical modification of the BF2
fragment with ethynylaryl modules has rarely been
attempted.[8]
Frequently encountered deficiencies in the use of Bodipy
derivatives are the small Stokes shifts (Dl ꢀ 600 cmꢁ1)
between the lowest energy absorption band and the emission
band, and the quenching of their fluorescence on conjugation
to proteins.[5] Larger Stokes shifts would be advantageous in
simplifying simultaneous excitation and emission detection,
since background interferences are reduced and wide-band
excitation and emission filters may be used, thus increasing
the intensity of the fluorescence signal. Also desirable is the
enhancement of this signal intensity by incorporation of a
chromophore active in the region 350–370 nm where Bodipy
species do not absorb, pyrene is an example of such a
chromophore.[9]
Herein, we describe an elegant method for increasing the
Stokes shift of Bodipy derivatives and minimizing fluores-
cence quenching after bioconjugation. This approach involves
functionalization of the Bodipy core by binding ethynylpyr-
ene entities directly to the boron center. Irradiation of the
pyrene chromophore results in exclusive emission from the
Bodipy core as a result of efficient intramolecular energy
[*] Dr. G. Ulrich, C. Goze, Dr. R. Ziessel
Laboratoire de Chimie Molꢀculaire, ECPM
25 rue Becquerel, 67087 Strasbourg Cedex 02 (France)
Fax: (+33)3-9024-2689
E-mail: gulrich@chimie.u-strasbg.fr
ziessel@chimie.u-strasbg.fr
Prof. M. Guardigli, Prof. A. Roda
Department of Pharmaceutical Sciences
University of Bologna
Via Belmeloro 6, 40126 Bologna (Italy)
[**] This work was supported by the Centre National de la Recherche
Scientifique, and the Ministꢁre de la Recherche et des Nouvelles
Technologies. “Cascatelle” is a French word meaning “a very small
cascade”.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200500808
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Angewandte
Chemie
transfer, so that these new dyes function as a
small cascade device or “cascatelle”. Further
functionalization enables ready attach-
ment of the dyes to biopolymers such as
proteins.
While trigonal alkynylboranes are well-
known,[10] tetrahedral species are rare[11] and
herein we describe the first such compounds
in which two ethynyl units are bound to the
boron center of a bora-indacene (Figure 1).
The syntheses of 1 and 2 involve the displace-
ment of fluoride from boron by 4-lithioethy-
nyltoluene
and
1-lithioethynylpyrene,
respectively. Both compounds are obtained
in satisfactory yield (1 62%, 2 30%), and
have been characterized by chemical analy-
ses, full spectroscopic measurements, and
crystal structure determinations.
The structure determinations (Figure 1)
show an almost exact tetrahedral geometry
for the sterically congested boron atoms. In
both, the N-B-N angle is 1068, while the C-B-
Figure 1. Structures of 1 (a) and 2 (b). Selected bond lengths [ꢂ] and angles [8]: for 1 B-C28 1.589,
B-C19 1.595, B-N1 1.562, B-N2 1.557, C28-C29 1.206, C19-C20 1.204, N1-C1 1.350, N1-C8 1.403,
N2-C17 1.351, N2-C11 1.401; C28-B-C19 114.76, C28-B-N2 108.67, N1-B-N2 106.05, N2-B-C19
109.62. For 2 B-C19 1.603, B-C37 1.585, B-N1 1.563, B-N2 1.565, C20-C19 1.187, C37-C38 1.194,
N1-C1 1.352, N1-C8 1.402, N2-C17 1.344, N2-C11 1.403; C19-B-C37 111.61, C19-B-N1 108.18,
N1-B-N2 105.89, N2-B-C37 109.64, N2-B-C19 110.99.
ꢁ
C angle for 1 is 1158 and for 2 is 1118. The B
ꢂ
C and C C bonds fall in the ranges 1.58–
1.60 ꢀ and 1.18–1.20 ꢀ, respectively. The B–
C distances are longer than those of trigonal
tris(3,3-dimethyl-1-butynyl)borane but similar to those in its
tetrahedral pyridine adduct.[12]
The electronic absorption spectra of 1 and 2 reflect the
presence of the separate Bodipy and ethynylaryl units
(Figure 2). A strong band at 516 nm is attributed to the S0!
S1 Bodipy transition, while a second weaker and broader band
at 371 nm (clearly seen in Figure 2a) is typical of the S0!S2
transition.[13,14] Peaks observed at higher energies may be
confidently assigned to the spin-allowed p–p* transitions of
the ethynyl (l ꢀ 320 nm) and toluyl (l ꢀ 265 nm) groups
(Figure 2). For 2, a strong absorption at 330–370 nm, partially
overlapping the S0!S2 Bodipy transition, is assigned to the
ethynylpyrene unit.[9] The spectroscopic data are summarized
in Table 1.
When excited at 516 nm, both 1 and 2 emit strongly, with
high fluorescence quantum yields, in the region 535–540 nm.
The radiative rate constants have values close to 1.5 ꢁ 108 sꢁ1.
For 2, excitation in the pyrene absorption band did not lead to
pyrene emission but instead to emission characteristic of the
indacene core. The fluorescence excitation spectrum matches
the absorption spectrum, indicating that there is an efficient
energy transfer from the pyrene to the Bodipy moiety. The
quantum yield measurements are consistent with an efficiency
close to 100% for this transfer. The result is that there are
virtual Stokes shifts of approximately 104 cmꢁ1.
To devise a means of grafting these highly fluorescent
units onto biopolymers, we synthesized an indacene deriva-
tive bearing an iodophenyl substituent on the meso position.
Substitution of the iodine by a carboxybutylethynyl unit
ultimately led to the activated ester 3d (Scheme 1), which was
expected to be reactive towards protein amino acid side-chain
nucleophiles such as the terminal amino group of lysine
(Scheme 1).[16]
Figure 2. Absorption (c), emission (a), and fluorescence excita-
tion spectra (g) for compounds 1 (a) and 2 (b) in dichloromethane
solution. Emission spectra were obtained upon excitation in the ethy-
nyltoluyl moiety absorption (252 nm) for 1 and in the ethynylpyrene
moiety absorption (371 nm) for 2, while fluorescence excitation spectra
were measured upon emission at 535 nm.
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Table 1: Spectroscopic[a] data at 298 K for the new compounds.
succimimide (NHS) esters.[17] Indeed, it
proved possible to attach multiple fluores-
cent tags to BSA using 3d in various label/
protein ratios (ranging from 2:1 to 10:1), in
a 1:1 (v/v) water/DMSO solution. Charac-
terization of the conjugates in terms of
label/protein molar ratio was performed by
means of UV/Vis absorption spectroscopy,
under the assumption that the absorption
spectra of the conjugates are the sum of the
absorption spectra of BSA and the Bodipy
dye and that these spectra coincide with
those of the free BSA and 3e, respectively
(Figure 3).
Compound labs [nm] emax [Mꢁ1 cmꢁ1
]
lF [nm] tF [ns]
f
F
[%] h[c] [%] kr [108 sꢁ1
]
knr [106 sꢁ1
]
[b]
1
2
516
516
371
523
370
522
370
67100
73000
95000
50000
61500
61000
88000
537
535
535
539
539
538
538
9.0
6.2
95
94
90
90
85
82
55
1.1
1.5
5.6
9.7
96
3b
3e
6.2
5.0
1.5
94
1.6
67
16.1
36.0
[a] Determined in aerated dichloromethane solution. [b] Determined using rhodamine 6G (fF =0.76 in
water[15]) as reference. All f were corrected for changes in refractive index. [c] Efficiency energy transfer
F
from ethynylpyrene to the indacene moiety, calculated by dividing the quantum yield found by excitation
in the chromophores by those found by excitation in the indacene moiety.
The experimentally determined label/
protein molar ratios of the conjugates were
in good agreement with the label/protein
molar ratio used in the conjugation reaction
(Table 2), indicating a high reaction yield.
These BSA conjugates essentially maintain
the spectroscopic characteristics of the
model compound 3e (Scheme 1 and
Table 1). The fluorescence emission of
aqueous solutions of the conjugates shows
maximum intensity at 544 nm (Figure 4),
being slightly red-shifted in comparison to
that of compound 3e in dichloromethane.
All the protein conjugates remain strongly
fluorescent, with an efficiency of energy
transfer from the ethynylpyrene chromo-
phore to the indacene moiety similar to that
found for 3e (see Figure 3). The efficiency
of the energy transfer from the pyrene to
=
Scheme 1. 1) HC C(CH2)4COOEt (1 equiv), [PdCl2(PPh3)2] (6% mol), CuI (10% mol), RT, 16 h, 91%.
2) NaOH (10 equiv), ethanol/THF (1:1), 608C, 12 h, 87%. 3) DMAP (2 equiv), EDCI (2 equiv), N-
hydroxysuccinimide (2 equiv), RT, 1 h, 54%. 4) n-propylamine, RT, 1 h, 74%. DMAP=dimethylamino-
pyridine, EDIC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
The demonstration of the suitability of derivative 3d for
protein labeling and the evaluation of the spectroscopic
properties of its protein conjugates were performed using
bovine serum albumin (BSA) as a model protein which has
59 lysine residues that can potentially react with N-hydroxy-
the indacene moiety drops for 3e and the labeled BSA
conjugates when compared to that of 3b, which has an ester
function. This drop is likely to be due to the presence of the
amide function in 3e and the labeled conjugates which
provides a non-radiative channel for the deactivation pro-
cess.[18]
The fluorescence quantum yield decreases slightly with
the increase in the degree of labeling of the conjugates,
Table 2: Label/protein molar ratios evaluated by UV/Vis spectroscopy
and fluorescence properties of the BSA conjugates of 3d in aqueous
solution.
Conjugate
Label/protein molar ratio[a]
fF [b] [%]
h
[c] [%]
2:1
5:1
10:1
1.9–1.8:1
4.3–5.2:1
8.9–9.9:1
75[d], 45[e]
63[d], 38[e]
49[d], 32[e]
61
61
67
[a] The values are the label/protein molar ratios of the conjugates
evaluated from the absorption at 528 nm and from the comparison of the
absorption of the conjugate at 280 nm with those of free BSA and 3d.
[b] Determined using rhodamine 6G (fF =0.76 in water[15]) as reference.
[c] Efficiency of the energy transfer from the ethynyltoluyl or ethynylpyr-
ene chromophore to the indacene moiety, calculated by dividing the
quantum yield found by excitation in the chromophores by those found
by excitation in the indacene moiety. [d] Upon excitation in the indacene
moiety absorption at 528 nm. [e] Upon excitation in the ethynylpyrene
chromophore absorption at 370 nm.
Figure 3. UV/Vis absorption spectra of the BSA conjugates of 3d in
water at a concentration of about 2.3ꢃ10ꢁ6 m and the BSA:3d ratios
indicated; for comparison, the absorption spectra of BSA in water and
of 3e in dichloromethane at the same concentration are also
shown (g).
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Chemie
UV spectral region, the BSA conjugates of 3d are more
efficient emitters than the fluorescein-labeled immunoglobu-
lin (Figure 5a). The fluorescence emission of the BSA
conjugates is stronger than that of the fluorescein-labeled
IgG even when the latter is measured using fluorescein-
specific excitation and emission filters (Figure 5b); the BSA
conjugates are only weakly fluorescent using fluorescein
filters because such filters do not match the absorption and
emission bands of 3d). This observation demonstrates that the
peculiar absorption and emission properties of 3d could be
successfully exploited also using the conventional excitation
sources used in fluorescence microscopy, such as mercury arc
lamps. Indeed, their most intense emission line of such lamps
(365–366 nm) is close to the maximum of the absorption band
of the ethynylpyrene chromophore, whereas fluorescein has
only a weak absorption at this wavelength.
Figure 4. Absorption (c) and emission (a) spectra of the 10:1
BSA conjugate of 3d in water solution recorded upon excitation in the
ethynylpyrene moiety absorption at 370 nm, and the fluorescence exci-
tation spectra (g) measured upon emission at 544 nm.
indicating a slight degree of self-quenching. However, the
overall fluorescence intensity of the conjugates, as evaluated
on the basis of their molar extinction coefficients and
fluorescence quantum yields, still increases with the degree
of labeling (Figure 5 and Table 2).
To compare the performance of the new Bodipy deriva-
tive with that of conventional fluorescent labels, fluorescence-
imaging experiments were conducted. Figure 5 shows fluo-
rescence images of spots containing the same amount of the
BSA conjugates of 3d or a fluorescein-labeled rabbit
immunoglobulin G (IgG) with a 2.3:1 fluorescein/antibody
labeling ratio (the fluorescein-labeled IgG and the 2:1
conjugate spots thus contain comparable amounts of fluo-
rescent label molecules). The comparison of the fluorescence
intensities of the spots indicates that, upon excitation in the
The key feature of the present molecular design is the
introduction of a supplementary chromophore linked by an
ethynyl bridge to a tetrahedral boron atom. Such “Bodipyr-
ene” dyes have three outstanding features: 1) relative ease of
synthesis; 2) very large Stokes shifts resulting from an
efficient, spin-allowed energy transfer from the excited
pyrene subunit to the emitting state of the indacene center
(a “cascatelle” process); 3) convenient functionalization to
introduce an activated ester group suitable for grafting the
dye to biopolymers. Very importantly, attachment of the dyes
to a protein appears to produce only minor quenching effects
and the singular other properties of the dyes are maintained.
Fluorescence-imaging microscopy shows that these new
labels are considerably superior to conventional fluorophores.
The notion of a cascatelle process, clearly substantiated by the
present work, is open to many avenues of development,
leading to possibilities such as the multichromatic patterning
of biomaterials using a single excitation source.
Received: March 4, 2005
Keywords: boron · fluorescence imaging · luminescence ·
.
protein labeling · Stokes shift
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