Water-soluble red-emitting distyryl-borondipyrromethene (BODIPY) dyes for biolabeling

Article abstract of DOI:10.1002/chem.201103613

A series of water-soluble red-emitting distyryl-borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water-solubilizing groups on opposite faces of the fluorescent core to reduce or c

Full text of DOI:10.1002/chem.201103613

FULL PAPER
DOI: 10.1002/chem.201103613
Water-Soluble Red-Emitting Distyryl-Borondipyrromethene (BODIPY)
Dyes for Biolabeling
Song-lin Niu,[a] Cꢀdrik Massif,[b] Gilles Ulrich,[a] Pierre-Yves Renard,[b, c, d]
Anthony Romieu,*[b, c] and Raymond Ziessel*[a]
Abstract:
red-emitting
A
series of water-soluble
distyryl-borondipyrro-
the BODIPY scaffold for conjugation
to amine-containing biomolecules/bio-
polymers. The optical properties of
these dyes were evaluated under simu-
lated physiological conditions (i.e.,
pH 7.5) or in pure water. The emission
wavelength (lmax) of these labels was
found in the 640–660 nm range with
quantum yields from modest to unpre-
cedentedly high values (4 to 38%).
The bioconjugation of these distyryl-
BODIPY dyes with bovine serum
albumin (BSA) and the monoclonal
antibody (mAb) 12A5 was successfully
performed under mild aqueous condi-
tions.
methene (BODIPY) dyes were de-
signed and synthesized by using three
complementary approaches aimed at
introducing water-solubilizing groups
on opposite faces of the fluorescent
core to reduce or completely suppress
self-aggregation. An additional carbox-
ylic acid functional group was intro-
duced at the pseudo-meso position of
phosphate-buffered
saline
(PBS),
Keywords: biolabeling · BODIPYs ·
fluorescence · sulfonated linker ·
water soluble
Introduction
good water-solubility, and the presence of an additional
functional group for bioconjugation, have, however, to be
met.[3] This is a formidable challenge and only a limited
number of such labels belonging to the family of cyanine or
rhodamine dyes are currently commercially available.[4,5,6]
Recently, a new class of water-soluble red-emitting carbo-
pyronines displaying very attractive spectroscopic features
has also been studied.[6d]
The red to near-IR absorbing window of biological tissues is
typically in the 600–900 nm range. In this optical window,
the scattering and absorption of light by biological samples
and the background signal from cellular autofluorescence
(300–550 nm) are dramatically reduced.[1] Thus, red-absorb-
ing and emitting fluorophores tend to offer the best resolu-
tion and greatest contrast in biological labeling experiments
and the detection of various (bio)analytes either in vitro or
in vivo. In this context, the engineering and study of red-
emitting dyes are of great interest and applicability.[2] Sever-
al criteria, such as high fluorescence quantum yields (f)
under physiological conditions, high absorption coefficients
(e), relatively long lifetimes (1–5 ns), high photostability,
Unlike that of such fluorescent markers, the synthesis of
water-soluble and bioconjugable red-emitting borondipyrro-
methene dye (BODIPY) derivatives has been little studied,
despite their promising photophysical properties.[7] In 2006,
Atilgan and co-workers reported the first synthesis of
a water-soluble NIR fluorescent BODIPY dye by introduc-
tion of several PEG-type linkers both onto the 8-meso-
phenyl and 3,5-distyryl moieties of the BODIPY core.[8] Fur-
ther functionalization with a dipicolylamine-derived binding
unit provided a sensitive and selective ratiometric chemo-
sensor for ZnII ions.[9] However, the lack of an additional
functional group for bioconjugation excluded the use of
such water-soluble dyes as biolabeling reagents. More re-
cently, anionic and cationic substituted BF2-chelated tetra-
arylazadipyrromethene derivatives (aza-BODIPY), bearing
sulfonic acid, carboxylic acid, or quaternary amine moieties
have been synthesized.[10] However, the location of two
water-solubilizing groups on the same side of the aza-
BODIPY scaffold led to amphiphile-like species fluorescent
only in aqueous solutions containing additives that disrupt
aggregates.
[a] Dr. S.-l. Niu, Dr. G. Ulrich, Dr. R. Ziessel
Laboratoire de Chimie Molꢀculaire et Spectroscopies Avancꢀes
(LCOSA), LMSPC UMR 7515 au CRNS
Ecole Europꢀenne de Chimie, Polymꢁres et Matꢀriaux
25 rue Becquerel, 67087 Strasbourg Cedex 02 (France)
Fax: (+33)3-68-85-27-6
E-mail: ziessel@unistra.fr
[b] C. Massif, Prof. P.-Y. Renard, Dr. A. Romieu
COBRA-CNRS UMR 6014 & FR 3038
rue Lucien Tesniꢁre, 76131 Mont-Saint-Aignan (France)
Fax : (+33)2-35-52-29-71
E-mail: anthony.romieu@univ-rouen.fr
[c] Prof. P.-Y. Renard, Dr. A. Romieu
Universitꢀ de Rouen, Place Emile Blondel
76821 Mont-Saint-Aignan (France)
As an alternative, we developed two synthetic strategies
to introduce ionizable sulfonate groups onto BODIPY scaf-
folds and thereby improve their solubility in water and
aqueous buffers. The first protocol relied on the introduc-
[d] Prof. P.-Y. Renard
Institut Universitaire de France
103 Boulevard Saint-Michel, 75005, Paris (France)
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tion of polysulfonated pseudopeptidyl linkers derived from
a-sulfo-b-alanine onto the pseudo meso-position (functional-
ized with a carboxylic acid) by a post-synthetic Schotten–
Baumann amidification reaction.[11,12] The second approach
was based on the formation of polar zwitterions (sulfobe-
taine moieties) by quaternization of bis(dimethylpropargyl-
amine) BODIPY derivatives with 1,3-propanesultone.[13]
Both methods could be efficiently applied to green-emitting
BODIPY derivatives, causing them to become highly solu-
ble in water without affecting their photophysical properties.
However, our first attempts to introduce (a-sulfo-b-ala-
nine)-type linkers onto the pseudo meso-8-position of a red-
emitting distyryl-BODIPY scaffold led to fluorescence-
quenching of the resulting water-soluble dyes in aqueous
buffers due to the formation of aggregates.[14]
ed into the corresponding active N-hydroxysuccinimidyl
(NHS) ester for bioconjugation to amine-containing biomol-
ecules/biopolymers.[3] Interestingly, this NHS ester derivative
also offers a third anchoring point for a polysulfonated pseu-
dopeptidyl linker designed to improve the dyeꢃs water-solu-
bility and resistance to self-aggregation. In addition to the
practical implementation of these different water-solubiliz-
ing methodologies, the application of these new dyes in bio-
labeling has been demonstrated through direct conjugation
to a model protein bovine serum albumin (BSA) and a
monoclonal antibody (12A5).
Results and Discussion
Herein, we wish to report an alternative and more sophis-
ticated synthetic method to produce series of water-soluble
BODIPY derivatives that retain their strong red emission in
aqueous environments and are suitable for biolabeling appli-
cations. The basic concept was the introduction of several
water-solubilizing groups on opposite faces of the distyryl-
BODIPY scaffold through three complementary synthetic
approaches.[15] The first involves the formation of the sulfo-
betaine moieties through quaternization of dimethylamino
groups previously introduced on the styryl arms. The second
approach consists of the substitution of the fluorine atoms
of BODIPY with the appropriate ethynyl Grignard reagent.
In this approach, short PEG-type linkers or sulfobetaine
moieties are introduced onto the boron atom as the water-
solubilizing groups. Such functionalization of the distyryl-
BODIPY involves not only improving the dyeꢃs water-solu-
bility, but introduces an additional functional group for con-
jugation to biomolecules. Thus, an ethyl ester group was in-
troduced by a Pd-catalyzed carboalkoxylation reaction with
an aromatic halide group in the pseudo meso-position of the
BODIPY dye, and subsequent saponification gave the car-
boxylic acid derivative. The carboxylic acid can be convert-
Synthesis of water-soluble distyryl-BODIPY dyes: First, the
benzaldehyde derivative A was prepared in quantitative
yield from 4-bromobenzaldehyde and dimethylaminopro-
pyne by using a Pd0-promoted cross-coupling reaction under
mild conditions (Scheme 1). Then, a Knoevenagel condensa-
tion reaction between iodophenyl tetramethyl-BODIPY[16]
1 and 4-[3-(dimethylamino)-1-propyn-1-yl]benzaldehyde (A)
in a mixture of dry toluene and piperidine gave the key blue
distyryl derivative 2 in 33% yield after careful column chro-
matography. Then, the fluorine atoms on the boron center
were substituted by reaction with the Grignard reagent of
an ethyleneglycol (EG) chain or dimethylamino propyne,
leading to the corresponding compounds 3 and 4 in yields of
70 and 92%, respectively. Carboalkoxylation[17] promoted by
Pd0 efficiently converted the meso-iodine to the correspond-
ing ethyl ester group and gave the functionalized distyryl-
BODIPY derivatives 5–7.
Subsequently, quaternization of the dimethylpropargyl-
amine moieties with 1,3-propanesultone was performed in
an anhydrous organic solvent (DMF or 1,2-dichloroethane)
at 608C to afford sulfobetaine derivatives 8–10. NMR spec-
troscopy and mass spectral data are in full agreement with
the assigned molecular structures. For example, the well-re-
solved 1H NMR spectrum of 10 is shown in Figure 1. It
clearly exhibits all the characteristic signals of this sulfonat-
ed distyryl-BODIPY compound. The integration of eight
protons at d=7.84 ppm corresponds to the phenyl subunits
on the distyryl arms; the four protons observed at d=8.31
and 7.64 ppm belong to the AB system of the phenyl at the
pseudo-meso position. The quaternary dimethylammonium
groups give signals at d=3.35 and 3.04 ppm integrating for
12H in total. The four protons at d=2.39 and 2.14 ppm are
assigned to the CH2 groups of the sulfobetaine residues on
the styryl arms and on the boron center, respectively. In ad-
dition, the observed 16 Hz proton–proton coupling constant
is in agreement with the E configuration for the double
bonds, already reported for this type of Knoevenagel con-
densation on BODIPY dyes.[18] Since compound 10 with
four sulfobetaine groups was found to be very soluble in
water, no further sulfonation of its meso-phenyl substituent
was required. Thus, this compound was only subjected to an
alkaline hydrolysis to generate the bioconjugatable carbox-
Abstract in French: De nouveaux colorants fluorescents ꢀ ar-
chitecture distyrylborondipyrromethene (BODIPY), ꢁmettant
dans le rouge et solubles dans lꢂeau, ont ꢁtꢁ prꢁparꢁs en utili-
sant trois mꢁthodes efficaces de fonctionnalisation chimique
qui permettent lꢂintroduction de groupements hydrophiles
(motifs pseudo-PEG et sulfonate) sur les parties nord et sud
de leur squelette conjuguꢁ, et ce afin de rꢁduire ou de suppri-
mer totalement lꢂagrꢁgation en solution aqueuse. La solubilitꢁ
dans lꢂeau des fluorophores ainsi obtenus est ꢁlevꢁe et leurs
propriꢁtꢁs optiques en milieu physiologique remarquables et
comparables ꢀ celles dꢁterminꢁes dans les solvants organi-
ques pour les composꢁs hydrophobes parents. De plus, la prꢁ-
sence dꢂune fonction acide carboxylique en position mꢀso de
ces composꢁs distyryl-BODIPY a permis le marquage cova-
lent de protꢁines dꢂintꢁrÞt (albumine de sꢁrum bovin et anti-
corps monoclonaux 12A5) via la formation de liens amide
dans des conditions biocompatibles.
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
Scheme 1. Synthesis of water-soluble distyryl-BODIPY dyes 11–13 (TEA=triethylamine).
ylic acid 13 in a 23% isolated yield after flash column chro-
matography on a RP-C18 silica gel column.
three steps. Their structures were confirmed by ESI mass
spectrometry measurements and their purity was checked by
RP-HPLC.
In contrast, the bis(sulfobetaine) derivatives 8 and 9 were
found to be poorly water-soluble and a further Schotten–
Baumman amidification reaction with dipeptidyl linker (a-
sulfo-b-alanine)2 was therefore applied. After saponification
of the ethyl ester, these compounds were quantitatively con-
verted into the corresponding NHS ester by treatment with
the peptide coupling uronium reagent O-(N-succinimidyl)-
1,1,3,3-tetramethyl uronium tetrafluoroborate (TSTU) and
N,N-diisopropylethylamine (DIEA) in dry N-methyl-2-pyr-
rolidone (NMP).[19] Thereafter, the crude NHS ester was
subjected to aminolysis with the dipeptide (a-sulfo-b-ala-
nine)2 in aqueous NaHCO3 (pH 8.5) to give the water-solu-
ble distyryl-BODIPY dyes 11 and 12. RP-HPLC purification
by using aqueous triethylammonim bicarbonate (TEAB,
pH 7.5) buffer and acetonitrile as eluents, followed by de-
salting on Dowex H+ ion-exchange resin provided 11 and 12
in pure forms with an overall yield of 30–40% for the last
We assumed that the low overall yield for the synthesis of
13 was due to the chemical instability of the ethynylaryl sub-
stituents. Thus, we decided to construct the styryl arms of
the BODIPY core by using the 4-[(dimethylamino)methyl]-
benzaldehyde (B) instead of the 4-[3-(dimethylamino)-1-
propyn-1-yl]benzaldehyde derivative (A) as the starting ma-
terial. An original three-step synthetic protocol aimed at
preparing this unusual aldehyde was developed as described
in Scheme 2. First, the radical bromination of 4-iodotoluene
gave the 4-iodobenzyl bromide in 46% yield. Subsequent
nucleophilic substitution with dimethylamine in THF afford-
ed the N-(4-iodobenzyl)-N,N-dimethylamine in 93% yield.
Finally, a Pd0-promoted formylation reaction provided the
targeted 4-[(dimethylamino)methyl]benzaldehyde (B) in
a good 73% yield.
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R. Ziessel, A. Romieu et al.
Figure 1. 1H NMR spectrum (400 MHz) of sulfonated distyryl-BODIPY dye 10 recorded in a CD3OD/D2O mixture. Despite the solubility of this com-
pound in water, CD3OD was added to get a better resolved spectrum.
dye 18 (overall yield: 16% against 3% for dye 13). The
1H NMR spectrum of 18 is shown in Figure 2 and supports
the molecular structure of this free acid distyryl-BODIPY
dye.
Photophysical properties: The red-emitting distyryl-
BODIPY dyes 11–13 and 18 were found to be readily solu-
ble in water and related aqueous buffers in the concentra-
Scheme 2. Synthesis of 4-[(dimethylamino)methyl]benzaldehyde (B).
tion range (1 mm to 10 mm) largely suitable for biolabeling
applications. Their optical properties were evaluated under
This novel benzaldehyde derivative was used in the well-
established Knoevenagel condensation reaction involving 1,
to give the distyryl-BODIPY 14 (see Scheme 3 for the syn-
thesis of BODIPY dye 14) in a 40% yield. Subsequent sub-
stitution of the fluorine atoms by the alkynyl-Grignard re-
agent from N,N-dimethylaminopropyne gave dye 15 in 81%
yield. Thereafter, a Pd-catalyzed carboalkoxylation reaction
provided the ethyl ester 16 and quaternization of the terti-
ary amino groups by treatment with an excess of 1,3-pro-
panesultone in dry DMF provided the tetra-sulfonated de-
rivative 17, which was purified by flash column chromatog-
raphy on a RP-C18 silica gel column. Finally, saponification
of the ethyl ester was performed under standard conditions
to give the water-soluble and bioconjugatable distyryl-
BODIPY dye 18. Thus, a significantly higher overall yield
was obtained for this five-step synthesis relative to that in
the preparation of the first tetrakis(sulfobetaine) BODIPY
simulated physiological conditions (i.e., phosphate buffered
saline (PBS), pH 7.5) or pure water and the corresponding
data are collected in Table 1. In aqueous solution, dye 11,
despite the presence of a disulfonated tail and two sulfo-
betaine residues on the distyryl-BODIPY scaffold, displays
unusual features, with a dual absorption band, a lowered in-
tensity of the main p–p* absorption, and a broadening of all
absorption bands (Figure 3). Dye 11 shows a low absorption
coefficient (ꢀ20000mꢁ1 cmꢁ1) and a low fluorescence quan-
tum yield (4%; Table 1, entry 6). The dual absorption, the
breadth of the absorption bands, and the hypsochromic shift
of one to 601 nm is in keeping with the formation of non-
emissive aggregates.[14,20] Interestingly, the addition of BSA
protein (5% (w/v) in PBS) led to an increase of the quan-
tum yield to 20% (Figure 4). This protein is known to en-
hance the emission of many fluorophores due to a combina-
tion of rigidization, reduction in the polarity of the dyeꢃs mi-
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
Scheme 3. Synthesis of the water-soluble distyryl-BODIPY dye 18.
Figure 2. 1H NMR spectrum of sulfonated distyryl-BODIPY dye 18 recorded in CD3OD/D2O (300 MHz). Despite the solubility of this compound in
water, CD3OD was added to get a better resolved spectrum.
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R. Ziessel, A. Romieu et al.
Table 1. Spectroscopic properties of BODIPY dyes studied in this work. Data for the
less energetic absorption band.
croenvironment (binding in the hydrophobic BSA
pocket), and deaggregation.[21a] Moreover, PBS sol-
utions containing 5% BSA are often used as
a model for body fluids.
Compound
labs
e
lem
f
t
kr
[107 sꢁ1
knr
[107 sꢁ1
]
[c]
[d]
[nm]
[mꢁ1 cmꢁ1
]
[nm] [%][a] [ns][b]
E
]
N
2 (CH2Cl2)
3 (CH2Cl2)
4 (CH2Cl2)
5 (CH2Cl2)
7 (CH2Cl2)
11 (PBS)
12 (PBS)
13 (PBS)
14 (CH2Cl2)
15 (1,4-dioxane) 634
16 (1,4-dioxane) 634
646
647
648
646
647
110600
108500
92200
122200
83500
661 46
659 47
661 26
659 43
663 30
5.2
4.8
5.2
4.6
4.8
–
8.8
9.8
5.0
9.3
6.3
–
10.4
11.0
14.2
12.4
14.6
–
–
27.8
9.2
11.9
12.1
20.6
14.4
15.9
The substitution of both fluorine atoms at the
boron center by EG chains dramatically improves
the spectroscopic properties of the distyryl-
BODIPY dye under physiological conditions. Thus,
in aqueous solution, the optical properties of 12 in
the red region are quite similar to those of standard
distyryl-BODIPY derivatives in organic solvents
(Table 1).[22] The absorption spectrum of the dye
has its lowest energy absorption maximum centered
at 642 nm, corresponding to the S0!S1 (0–0) transi-
tion of the BODIPY core, with an absorption coef-
ficient of 55100mꢁ1 cmꢁ1 (Figure 5). The shoulder
centered at about 590 nm can be assigned to the
0–1 vibrational band of the same transition.[23] The
strong absorption band with an absorption coeffi-
cient of 76800mꢁ1 cmꢁ1 centered at 366 nm can
probably be assigned to the
601, 649 17900, 19300 655
4
642
641
632
55100
58000
64500
81300
84200
76600
92000
71000
657 22
657 25
640 40
645 25
649 36
643 30
646 35
641 38
–
–
2.7
6.5
6.3
5.3
3.4
4.5
3.9
9.3
6.2
4.0
6.8
8.8
7.8
9.7
17 (H2O)
17 (EtOH)
18 (H2O)
630
636
628
[a] Standard used for quantum yield measurements: cresyl violet in EtOH (f=51%),
lexc =578 nm[32] for 2–7 and 14–18, and sulfoindocyanine dye Cy 5.0 in PBS (f=20%),
lexc =600 nm[5] for 11–13. All f are corrected for changes in refractive index. [b] Life-
time. [c] kr =f/t. [d] knr =(1ꢁf)/t, assuming that the emitting state is produced with
unit quantum efficiency.
p!p* transition of the styryl
moieties.[24]
In emission, in contrast to the
BODIPY dye 11, the boron-
EG-substituted BODIPY dye
12 exhibits a strong fluores-
cence emission centered at
657 nm, with
a fluorescence
quantum yield of 22%. The ab-
sence of aggregates was con-
firmed by recording the excita-
tion spectrum, which perfectly
matches the absorption spec-
trum. In comparison with 11,
the better quantum yield and
the absence of aggregation of
12 indicate that the substitution
of fluorine atoms at the boron
center by bulky polar functional
groups can efficiently prevent
the aggregation of the distyryl-
BODIPY dye in aqueous
media. This fact was also con-
firmed with tetrakis(sulfobe-
taine) BODIPY dyes 13 and 18
(Table 1 and Figures 6 and 7).
Indeed, the introduction of
four sulfobetaine residues onto
one face of the BODIPY scaf-
fold totally overcomes the
water-solubility issues with the
distyryl-BODIPY dyes, remov-
ing the need for the introduc-
tion of a further polysulfonated
tail onto the pseudo meso-posi-
tion. The optical properties of
Figure 3. Absorption and emission (lexc. =600 nm) spectra of dye 11 in PBS at 258C. c: Absorption; c:
emission; c: excitation.
Figure 4. Normalized absorption and fluorescence emission (lexc =600 nm) spectra of 11 in PBS+5% (w/v)
BSA at 258C. c: Absorption; c: emission; c: excitation.
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
352 nm. In fluorescence, the
dye exhibits an emission maxi-
mum at 641 nm with a good
fluorescence quantum yield of
38%. Once again, the absorp-
tion, emission, and excitation
spectra confirm the absence of
aggregation in water. In addi-
tion, slight hypsochromic shifts
of about 14 nm were observed
in both the absorption and
emission maxima of dye 18 rel-
ative to those of the structural
analogue 13, due to the reduced
p-conjugation of the BODIPY
core.
Figure 5. Absorption and emission (lexc =600 nm) spectra of water-soluble BODIPY dye 12 in PBS at 258C.
c: Absorption; c: emission; c: excitation.
Bioconjugation: Since the most
emissive
water-soluble
BODIPY dyes, 12 and 13, are
functionalized with a free car-
boxylic acid group, their ability
to label proteins and antibodies
through reactions of their
active ester with the NH2
groups of e-lysine residues pres-
ent in these biopolymers was
assessed. BSA and the mono-
clonal antibody (mAb) 12A5
that recognizes the influenza
hemagglutinin (HA) epitope
tag[25] were chosen as the pro-
tein and the antibody, respec-
tively, for these bioconjugation
experiments.
To compare the labeling per-
formances of these novel red-
fluorescent markers with those
of classic CyDye reagents, simi-
lar reactions were performed
Figure 6. Absorption, excitation (lem =680 nm), and emission (lexc =600 nm) spectra of dye 13 in PBS at 258C.
c: emission; c: Absorption; c: excitation.
13 are quite similar to those of the dye 12 (Figure 6). In the
UV/Vis absorption spectrum, the lowest energy absorption
maxima is centered at 641 nm, with an absorption coeffi-
cient of 58000mꢁ1 cmꢁ1. The strong absorption centered at
369 nm can probably be assigned to the p!p* transitions of
the styryl groups. In fluorescence, the dye exhibits a strong
emission maximum centered at 657 nm with a fluorescence
quantum yield of 25%. The absorption, emission, and exci-
tation spectra of the dye confirm the absence of aggregation
in aqueous solution.
The optical properties of 18 were also evaluated in pure
water (Figure 7 and Table 1). In aqueous solution, the car-
boxylic acid 18 shows similar optical properties to those of
12 and 13. In the UV/Vis absorption spectrum, the lowest
energy absorption maximum is centered at 628 nm with an
absorption coefficient of 71000mꢁ1 cmꢁ1. The strong absorp-
tion of the p!p* transitions of the styryl groups appears at
with sulfoindocyanine dye Cy 5.0[5] under the same experi-
mental conditions. To avoid possible harmful effects of or-
ganic solvents and additives towards the mAb (or protein)
structure and functional activity, a carboxylic acid activation
protocol performed in deionized water and involving the
use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) and sulfo-NHS instead of TSTU/DIEA in a polar
aprotic solvent, such as DMF or DMSO, was preferred.[26] In
this case, the one-pot procedure without isolating the corre-
sponding sulfo-NHS ester of the BODIPY dye was pre-
ferred because these labeling reactions were typically per-
formed on a small scale (<1 mg of dye). However, isolation
of the active ester of the BODIPY dye can be performed by
RP-HPLC if necessary.
BSA and anti-HA mAb were labeled through overnight
incubation with a 13- and 31-fold molar excess of BODIPY
derivatives 12 and 13, respectively, in PBS (pH 7.5). The re-
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R. Ziessel, A. Romieu et al.
unchanged (Figures 8 and 9). In
addition, the conjugated dye 13
shows a more pronounced ten-
dency to form aggregates, as re-
vealed by the enlargement of
these absorption bands and the
blueshift of one of these bands
to 588 nm. The lesser tendency
of the conjugated dye 12 to
form nonemissive aggregates is
probably
a
further positive
effect of the disulfonated tail
grafted onto the meso-phenyl
substituent. The observed con-
jugation-induced diminution in
fluorescence efficiency is not
surprising and is attributed to
a combination of effects, with
Figure 7. Absorption, excitation (lem =680 nm), and emission (lexc =600 nm) spectra of water-soluble BODIPY
dye 18 in deionized water at 258C. c: Absorption; c: emission; c: excitation.
Table 2. Spectral properties in PBS of the proteins labeled with water-
soluble red-emitting dyes 12, 13, or Cy 5.0 and fluorophore to protein
molar ratios (F/P).
sulting protein fluorescent conjugates were purified by size-
exclusion chromatography on a Sephadex G-25 column.
Table 2 contains the absorption/emission wavelengths and
quantum yields of fluorescent proteins in PBS, together with
the attached fluorophore/protein molar ratios (F/P). The
F/P ratio was calculated from the absorbance of the fluores-
cent protein conjugate at 280 nm (corrected for BODIPY/
cyanine dye contribution) and the integral absorption of the
dyeꢃs longest wavelength absorption band. Use of the inte-
gral absorption instead of the commonly employed molar
absorption coefficient at the dyeꢃs absorption maximum is
more appropriate for the calcu-
Conjugated fluorescent dye
labs [nm][a]
lem [nm]
f [%][b]
F/P
mAb-12
BSA-12
mAb-13
BSA-13
mAb-Cy 5.0
BSA-Cy 5.0
370, 603, 650
372, 603, 651
370, 588, 650
371, 588, 650
610, 650
657
657
659
658
667
663
7
6
5
5
12
3
6.6
1.5
4.5
0.8
3.1
1.0
609, 651
[a] The absorption signal arising from the protein at about 280 nm is
omitted. [b] Determined at 258C by using sulfoindocyanine dye Cy 5.0
(FF =20% in PBS at 258C) as a standard.[5]
lation of F/P ratios of conju-
gates showing conjugation-in-
duced changes in the shape of
the fluorophoreꢃs absorption
band.[21]
The F/P values achieved with
the meso-substituted BODIPY
dye 12 were significantly higher
than those achieved with the
dye 13 and Cy 5.0, which sug-
gests a greater reactivity of its
sulfo-NHS ester. Indeed, the
added (a-sulfo-b-alanine)dipep-
tidyl spacer enables projection
of the bioconjugable CO2H
group further away from the
sterically hindered distyryl-
Figure 8. Normalized absorption and emission (lexc =600 nm) spectra of conjugate BSA–12 in PBS at 258C.
c: Absorption; c: emission; c: excitation.
BODIPY core than in 13, thus
ensuring a better capability for
the acylation of primary amines
within proteins, especially under pH-neutral conditions.
When compared to the noncovalently bound water-solu-
ble distyryl-BODIPY dyes 12 and 13, the absorption
maxima of the fluorescent protein conjugates are slightly
redshifted by about 8 nm, whereas emission maxima remain
the main contributions arising from the formation of non-
fluorescent aggregates (H-type homodimers) and from fluo-
rescence energy resonance transfer (FRET) from the fluo-
rescent monomeric dyes to the increasing number of non-
emissive dimers on the biopolymers.[21a] Nonetheless, these
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
Experimental Section
General remarks: Column chromatog-
raphy purifications were performed
either on silica gel 60m (40–63 mm)
from Macherey–Nagel or on alumi-
num oxide 90 standardized from
Merck. Reversed-phase flash column
chromatography was performed on
octadecyl-functionalized
silica gel
(mean pore size 60 ꢄ, 37–74 mm) from
Aldrich. TLC analyses were carried
out on Macherey–Nagel Alugram Sil
G/UV254 aluminum sheets. All reac-
tions were performed under a dry at-
mosphere of argon by using standard
Schlenk tube techniques. All chemicals
were used as received from commer-
cial sources without further purifica-
tion unless otherwise stated. All sol-
vents were dried by following standard
procedures (CH2Cl2: distillation over
P2O5, C2H4Cl2: distillation over P2O5,
diethyl ether: distillation over Na0/
Figure 9. Normalized absorption and emission (lexc =600 nm) spectra of conjugate BSA–13 in PBS at 258C.
c: Absorption; c: emission; c: excitation.
red labels maintain moderate fluorescence quantum yields
upon bioconjugation and are expected to have utility in flu-
orescent biosensing applications, especially those involving
the design of activatable fluorescent probes.[27]
benzophenone, DMF: distillation over BaO, THF: distillation over Na0/
benzophenone, toluene: disillation over Na0). NMP (peptide synthesis
grade) was purchased from Iris Biotech GmbH. The HPLC-gradient
grade acetonitrile (CH3CN) and methanol (CH3OH) were obtained from
VWR. PBS (100 mm phosphate+150 mm NaCl, pH 7.5) and aqueous
mobile phases for HPLC were prepared by using water purified with
a Milli-Q system (purified to 18.2 MWcm). 4-Iodobenzyl bromide,[28]
N-(4-iodobenzyl)-N,N-dimethylamine,[29]
[PdCl
N
and
Conclusion
[Pd
AHCTUNGTRENNUNG
nated linker a-sulfo-b-alaninyl-a-sulfo-b-alanine was prepared from
b-alanine by using synthetic procedures recently reported by us.[12] Sulfo-
indocyanine dye Cy 5.0 (also named Cy 5.29) was prepared by using a lit-
erature procedure.[5]
In this contribution, four novel water-soluble and bioconju-
gable red fluorophores based on a distyryl-BODIPY scaf-
fold were synthesized and studied. Our synthetic approach
is based on the introduction of water-solubilizing groups
(sulfonic acid and sulfobetaine moieties) at the final stages
of the BODIPY synthesis, through efficient alkylation and/
or acylation of preintroduced functional reactive groups
(tertiary amine and carboxylic acid). Thus, the time-consum-
ing handling and purification (by RP-HPLC or RP-C18 flash
column chromatography) of highly polar fluorescent materi-
als is advantageously limited to the final product. We found
that the fluorescence quantum yield of these distyryl-
BODIPY dyes in aqueous solution is primarily dependent
on the substitution pattern of their boron atom, which also
determines the aggregation behavior. Sterically demanding
subsitutents on the boron center and negatively charged sul-
fonate groups dramatically reduce the tendency to aggre-
gate, thereby leading to bright fluorophores with quantum
yields in the 22–38% range. To our knowledge, these are
the highest values reported to date for red-emitting
BODIPY dyes under physiological conditions. These new
dyes were also tested for protein labeling and the conjugates
obtained display satisfactory fluorescence properties. With
compounds 12, 13, and 18, we have established attractive
new diagnostic tools for in vitro and in vivo fluorescence ap-
plications.
Instruments and methods: Ion-exchange chromatography (for desalting
water-soluble BODIPY dyes) was performed with an Econo-Pac Dispos-
able chromatography column (Bio-Rad, #732–1010) filled with an aque-
ous solution of Dowex 50WX8–400 (Alfa Aesar, ꢀ7 g for 10 mg of dye,
15ꢅ50 mm bed), regenerated by using a 10% HCl aqueous solution and
equilibrated with deionized water. Size-exclusion chromatography (for
purification of fluorescently labeled proteins) was performed with an
Econo-Pac Disposable chromatography column (Bio-Rad, #732–1010)
filled with an aqueous solution of Sephadex G-25 Fine (Amersham Bio-
sciences AB, 15ꢅ40 mm bed), equilibrated with PBS (0.01m phosphate,
0.015m NaCl, pH 7.5). 1H- and 13C NMR spectra were recorded on
Bruker AC 200, Avance 300, and Avance 400 spectrometers working at
200, 300, or 400 MHz, respectively. Chemical shifts are expressed in parts
per million (ppm) from the residual nondeuterated solvent signal. J
values are expressed in Hz. Analytical HPLC was performed on
a Thermo Scientific Surveyor Plus instrument equipped with a PDA de-
tector. Semi-preparative HPLC was performed on a Thermo Scientific
SPECTRASYSTEM liquid chromatography system (P4000) equipped
with a UV/Vis 2000 detector. MS (ESI) were obtained either with a Finni-
gan LCQ Advantage MAX (ion trap) apparatus or a JEOL JMS-T100
LO Acc TOF. Elemental analysis was conducted by an Elementar vario
MICRO Cube.
HPLC separations: Four chromatographic systems were used for the ana-
lytical experiments and purification steps.
System A: RP-HPLC (Thermo Hypersil GOLD C18 column, 5 mm, 4.6ꢅ
100 mm) with CH3CN and trifluoroacetic acid (aqueous TFA, 0.1%,
pH 2.2) as eluents (20% CH3CN (5 min), followed by a linear gradient
from 20 to 100% (32 min) of CH3CN) at a flow rate of 1.0 mLminꢁ1
Triple UV/Vis detection was achieved at 254, 530, and 640 nm.
.
System B: Semi-preparative RP-HPLC (Varian Kromasil C18 column,
10 mm, 21.2ꢅ250 mm) with CH3CN and 0.1% aqueous TFA as eluents
Chem. Eur. J. 2012, 00, 0 – 0
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R. Ziessel, A. Romieu et al.
(10% CH3CN (10 min), followed by a linear gradient from 10 to 100%
(45 min) of CH3CN) at a flow rate of 16.0 mLminꢁ1. Dual visible detec-
tion was achieved at 366 and 635 nm.
787.2; found: 788.2 [M+H]+ (100); elemental analysis calcd (%) for
C43H40BF2IN4: C 65.50, H 5.11, N 7.11; found: C 65.22, H 4.64, N 6.82.
Compound 3:
A solution of 3-(2-methoxyethoxy)-1-propyne (68 mg,
System C: System A with aqueous triethylammonium acetate (TEAA,
100 mm, pH 7.0) instead of 0.1% aqueous TFA.
0.60 mmol) in dry THF (5 mL) was placed in a Schlenk tube under an
argon atmosphere. A 1.0m solution of EtMgBr in THF (0.52 mL) was
then added and the reaction mixture was stirred at 608C for 1 h. The re-
sulting anion was then transferred through a cannula to a solution of dis-
tyryl-BODIPY 2 (120 mg, 0.15 mmol) in dry THF (3 mL) previously
placed in a Schlenk tube under an argon atmosphere. The resulting reac-
tion mixture was stirred at 608C for about 15 min, until the complete
consumption of the starting BODIPY was observed by TLC analysis.
Then, deionized water (3 mL) was added. The mixture was then dissolved
in CH2Cl2 and sequentially washed with deionized water and brine. The
organic layer was dried over anhydrous MgSO4 and evaporated to dry-
ness. The crude product was purified by chromatography on a silica gel
column with the following eluent (CH2Cl2/CH3OH 93:7) to afford bis-
System D: Semi-preparative RP-HPLC (Varian Kromasil C18 column,
10 mm, 21.2ꢅ250 mm) with CH3CN and aqueous triethylammonium bi-
carbonate (TEAB 50 mm, pH 7.5) as eluents (5% CH3CN (10 min), fol-
lowed by a linear gradient from 5 to 100% (47 min) of CH3CN) at a flow
rate of 16.0 mLminꢁ1. Dual visible detection was achieved at 366 and
640 nm.
Spectroscopic measurements: UV/Vis spectra were recorded by using
a Shimadzu UV-3600 dual-beam grating spectrophotometer with a 1 cm
quartz cell. Fluorescence spectra were recorded on a HORIBA Jobin–
Yvon fluoromax 4P spectrofluorimeter. All fluorescence spectra were
corrected. The following equation was used to determine the relative
fluorescence quantum yield was determined by using Equation (1) in
which A is the absorbance at the excitation wavelength (in the range
0.01–0.1 A.U.), F is the area under the corrected emission curve, n is the
refractive index of the solvents (at 258C) used in measurements, and the
subscripts S and X represent standard and unknown, respectively. The
AHCTUNGTRENNUNG
=
8.0 Hz, 4H), 7.48 (d, JHH =8.1 Hz, 4H), 7.13 (m, 4H), 6.66 (s, 2H), 4.15
(s, 4H), 3.56 (s, 4H), 3.49 (m, 4H), 3.20 (s, 6H), 3.16 (m, 4H), 2.43 (s,
12H), 1.47 ppm (s, 6H); 13C NMR (CDCl3, 75 MHz): d=152.0, 140.7,
138.5, 137.0, 135.1, 133.8, 132.5, 130.7, 127.4, 123.4, 121.8, 118.7, 92.1,
86.0, 68.5, 59.0, 48.7, 44.2, 29.9, 15.4 ppm; MS (ESI, positive mode): m/z
(%): calcd for C55H58BIN4O4: 975.4; found: 977.2 [M+H]+ (100);
elemental analysis calcd (%) for C55H58BIN4O4·H2O: C 66.40, H 6.08,
N 5.63; found: C 66.12, H 5.74, N 5.44.
following standards were used: Cresyl violet in ethanol (f=50%, lexc
546 nm or f=51%, lexc =578 nm) and Cy 5.0 in PBS (f=20%, lexc
=
=
600 nm).[5,32] Luminescence lifetimes were measured on an Edinburgh In-
struments spectrofluorimeter equipped with an R928 photomultiplier and
a PicoQuant PDL 800-D pulsed diode connected to a GwInstect GFG-
8015G delay generator. No filter was used for the excitation. Emission
wavelengths were selected by a monochromator. Lifetimes were decon-
Compound 4:
A solution of 1-dimethylamino-2-propyne (52 mg,
0.62 mmol) in dry THF (3 mL) was placed in a Schlenk tube under an
argon atmosphere. A 1.0m solution of EtMgBr in THF (0.435 mL) was
added and the reaction mixture was and stirred at 608C for 1 h. The re-
sulting anion was then transferred through a cannula to a solution of dis-
tryryl-BODIPY 2 (100 mg, 0.124 mmol) in dry THF (3 mL) previously
placed in a Schlenk tube under an argon atmosphere. The resulting reac-
tion mixture was stirred at 608C for 15 min until the complete consump-
tion of the starting BODIPY was observed by TLC analysis. Then, the
mixture was sequentially treated with deionized water and brine and ex-
tracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4
and evaporated to dryness. The crude product was purified by chroma-
tography on a Al2O3 column with the following eluent (CH2Cl2/CH3OH
voluted with FS-900 software by using
(LUDOX) for instrument response.
a
light-scattering solution
2
ð1Þ
ꢀðXÞ ¼ ðAS=AXÞðFX=FSÞðnX=nSÞ ꢀðSÞ
4-[3-(Dimethylamino)-1-propyn-1-yl]benzaldehyde
(A):
[PdACTHUNGTRENNU(G PPh3)4]
(0.36 g, 0.32 mmol) and 1-dimethylamino-2-propyne (0.67 g, 8.10 mmol)
were sequentially added to a degassed solution of 4-bromobenzaldehyde
(1.0 g, 5.40 mmol) in benzene (3 mL) and triethylamine (3 mL) under an
argon atmosphere. The resulting reaction mixture was stirred at 608C for
10 h until the complete consumption of the starting material was ob-
served by TLC analysis. The mixture was then evaporated and purified
by chromatography on a silica gel column with the following eluents
(CH2Cl2 100%, and then CH2Cl2/CH3OH 97:3) to afford the desired
compound (1.13 g, quant. yield). 1H NMR (CDCl3, 200 MHz): d=10.00
(s, 1H), 7.82 (d, JHH =8.3 Hz, 2H), 7.58 (d, JHH =8.3 Hz, 2H), 3.54 (s,
2H), 2.42 ppm (s, 6H); MS (ESI, positive mode): m/z (%): calcd for
C12H13NO: 187.1; found: 188.1 [M+H]+ (100); elemental analysis calcd
(%) for C12H13NO: C 76.98, H 7.00, N 7.48; found: C 76.64, H 6.82,
N 7.17.
97:3) to give bis
(CDCl3, 300 MHz): d=8.39 (d, JHH =16.4 Hz, 2H), 7.86 (d, JHH =8.2 Hz,
2H), 7.61 (d, JHH =8.2 Hz, 4H), 7.47 (d, JHH =8.0 Hz, 4H), 7.15 (d, JHH
(ethynyl) BODIPY 4 (114 mg, quant. yield). 1H NMR
ACHTUNGTRENNUNG
=
16.6 Hz, 2H), 7.11 (d, JHH =8.1 Hz, 2H), 6.67 (s, 2H), 3.53 (s, 4H), 3.20
(s, 4H), 2.42 (s, 12H), 2.20 (s, 12H), 1.47 ppm (s, 6H); 13C NMR (CDCl3,
75 MHz): d=151.8, 140.3, 138.2, 137.8, 136.8, 135.1, 133.4, 132.1, 131.8,
130.6, 127.2, 123.3, 122.0, 118.5, 94.8, 90.8, 86.5, 85.4, 48.9, 48.7, 44.3, 34.1,
29.7, 22.4, 15.2, 14.1 ppm; MS (ESI, positive mode): m/z (%): calcd for
C53H56BIN6: 913.4; found: 915.2 [M+H]+ (100); elemental analysis calcd
(%) for C53H56BIN6·H2O: C 66.95, H 6.36, N 8.84; found: C 66.78,
H 6.21, N, 8.64.
Compound 2: A mixture of 8-(4-iodophenyl)-1,3,5,7-tetramethyl-4,4-di-
fluoro-4-bora-3a,4a-diaza-s-indacene (1; 500 mg, 1.11 mmol) and benzal-
dehyde derivative A (415 mg, 2.22 mmol) was dissolved in dry toluene
(20 mL) and piperidine (0.5 mL). The resulting reaction mixture was
heated at 1408C in a Dean–Stark apparatus for 2 h. Thereafter, the
Dean–Stark receiver was removed and the mixture was heated with
argon bubbling until the solvent was completely removed. The resulting
crude product was treated with saturated aqueous NaHCO3 and deio-
nized water, and finally extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4 and evaporated to dryness. The crude resi-
due was purified by chromatography on a silica gel column with a step
gradient of CH3OH (3–7%) in CH2Cl2 as the mobile phase giving the dis-
tyryl-BODIPY 2 (193 mg, 22%). 1H NMR (CDCl3, 300 MHz): d=7.86
(d, JHH =7.7 Hz, 2H), 7.71 (d, JHH =16.3 Hz, 2H), 7.56 (d, JHH =7.9 Hz,
4H), 7.46 (d, JHH =7.8 Hz, 4H), 7.22 (d, JHH =16.4 Hz, 2H), 7.08 (d,
Compound 5: Distyryl-BODIPY 2 (100 mg, 0.12 mmol) was dissolved in
a
mixture of absolute EtOH (4 mL) and triethylamine (3 mL).
[PdCl2A(PPh3)2] (9 mg, 0.012 mmol) was added and the reaction mixture
CTHUNGTRENNUNG
was kept under a CO atmosphere for 4 h and heated at 608C. Thereafter,
the mixture was treated with saturated aqueous NaHCO3 and deionized
water and extracted with CH2Cl2. The organic layer was dried over anhy-
drous MgSO4. The solvent was then removed and the crude product was
purified by chromatography on silica gel column with the following
eluent (CH2Cl2/CH3OH 93:7) to afford the ethyl ester 5 in a pure form
(78 mg, 85%). 1H NMR (CDCl3, 300 MHz): d=8.18 (d, JHH =8.1 Hz,
2H), 7.70 (d, JHH =16.4 Hz, 2H), 7.54 (d, JHH =8.1 Hz, 4H), 7.43 (d,
J
HH =8.1 Hz, 4H), 7.42 (d, JHH =7.7 Hz, 2H), 7.20 (d, JHH =16.2 Hz, 2H),
6.62 (s, 2H), 4.41 (q, JHH =7.2 Hz, 2H), 3.51 (s, 4H), 2.39 (s, 12H), 1.42
(t, JHH =7.3 Hz, 3H), 1.40 ppm (s, 6H); 13C NMR (CDCl3, 75 MHz): d=
166.2, 152.9, 142.2, 139.9, 137.9, 136.4, 133.4, 132.3, 131.4, 130.5, 128.9,
127.6, 123.7, 120.0, 118.5, 86.3, 85.9, 61.6, 48.9, 46.4, 44.4, 15.0, 14.5 ppm;
MS (ESI, positive mode): m/z (%): calcd for C46H45BF2N4O2: 733.4;
J
HH =7.7 Hz, 2H), 6.65 (s, 2H), 3.52 (s, 4H), 2.40 (s, 12H), 1.48 ppm (s,
6H); 13C NMR (CDCl3, 75 MHz): d=152.9, 142.2, 138.6, 136.4, 135.9,
134.8, 133.5, 130.6, 127.6, 123.8, 119.9, 118.4, 95.1, 86.7, 85.7, 48.9, 44.5,
15.2 ppm; MS (ESI, positive mode): m/z (%): calcd for C43H40BF2IN4:
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
found: 735.4 [M+H]+ (100); elemental analysis calcd (%) for
C46H45BF2N4O2: C 75.20, H 6.17, N 7.62; found: C 74.89, H 5.92, N 7.45.
139.8, 138.5, 132.9, 131.5, 130.9, 130.1, 128.7, 127.2, 122.5, 119.9, 118.6,
91.8, 67.9, 63.1, 61.4, 59.1, 58.2, 55.3, 41.9, 18.7, 14.7, 13.9 ppm; MS (ESI,
positive mode): m/z (%): calcd for C64H75BN4O12S2: 1165.5; found:
1167.5 [M+H]+ (100); the compound was too hygroscopic for suitable el-
emental analysis.
Compound 6: Distyryl-BODIPY 3 (104 mg, 0.10 mmol) was dissolved in
a mixture of absolute EtOH (4 mL) and triethylamine (3 mL). [PdCl2-
ACHTUNGTRENNUNG(PPh3)2] (8 mg, 0.01 mmol) was added and the reaction mixture was kept
Compound 10: Distyryl-BODIPY 7 (88 mg, 0.102 mmol) was dissolved in
dry DMF (2 mL). 1,3-Propanesultone (250 mg, 2.05 mmol) was added
and the resulting reaction mixture was stirred at 608C for 10 h. Then, ad-
dition of EtOAc led to precipitation of the crude product, which was re-
covered by centrifugation. Purification by flash chromatography on
a RP-C18 silica gel column by using the following eluent (H2O/THF
under a CO atmosphere for 5 h and heated at 608C. Thereafter, volatiles
were removed and the resulting residue was purified by chromatography
on a silica gel column with the following eluent (CH2Cl2/CH3OH 93:7) to
afford the ethyl ester 6 in a pure form (116 mg, quantitative yield).
1H NMR (CDCl3, 300 MHz): d=8.24 (d, JHH =16.3 Hz, 2H), 8.19 (d,
J
HH =8.5 Hz, 2H), 7.58 (d, JHH =8,3 Hz, 4H), 7.48 (d, JHH =8.3 Hz, 4H),
1
85:15) afforded 10 in a pure form (60 mg, 43%). H NMR (CD3OD/D2O,
7.46 (d, JHH =7.9 Hz, 2H), 7.13 (d, JHH =16.5 Hz, 2H), 6.65 (s, 2H), 4.43
(q, JHH =7.2 Hz, 2H), 4.15 (s, 4H), 3.59 (s, 4H), 3.49 (m, 4H), 3.20 (s,
6H), 3.16 (m, 4H), 2.46 (s, 12H), 1.45 (t, JHH =7.3 Hz, 3H), 1.41 ppm (s,
6H); 13C NMR (CDCl3, 75 MHz): d=166.2, 152.1, 140.7, 140.3, 138.3,
132.5, 131.3, 130.4, 129.1, 127.4, 123.2, 121.9, 118.8, 92.1, 86.4, 85.4, 61.6,
59.6, 59.0, 48.7, 46.0, 44.1, 29.9, 15.2, 14.5, 8.8 ppm; MS (ESI, positive
mode): m/z (%): calcd for C58H63BN4O6: 921.5; found: 922.4 [M+H]+
(100); elemental analysis calcd (%) for C58H63BN4O6·H2O: C 74.03,
H 6.96, N 5.95; found: C 73.78, H 6.70, N 5.66.
400 MHz): d=8.31 (d, JHH =8.4 Hz, 2H), 8.22 (d, JHH =16.2 Hz, 2H),
7.84 (s, 8H), 7.58 (d, JHH =16.2 Hz, 2H), 7.04 (s, 2H), 4.66 (s, 4H), 4.50
(q, JHH =7.2 Hz, 2H), 4.36 (s, 4H), 3.82 (m, 4H), 3.50 (m, 4H), 3.35 (s,
12H), 3.04 (s, 16H), 2.70 (m, 4H), 2.39 (m, 4H), 2.14 (m, 4H), 1.56 (s,
6H), 1.50 ppm (t,
J
HH =7.20 Hz, 3H); 13C NMR (CD3OD/D2O,
100 MHz): d=153.0, 143.5, 139.1, 136.3, 134.5, 132.9, 131.6, 130.2, 128.6,
122.7, 120.5, 92.6, 79.2, 64.2, 63.8, 62.8, 56.6, 20.1, 15.2, 14.6 ppm; MS
(ESI, positive mode): m/z (%): calcd for C68H85BN6O14S4: 1347.5; found:
1349.6 [M+H]+ (100); the compound was too hygroscopic for suitable el-
emental analysis.
Compound 7: The same procedure as described above was applied to dis-
tyryl-BODIPY 4 (100 mg, 0.11 mmol). Purification of the crude product
by chromatography on an Al2O3 column with the following eluent
(CH2Cl2/CH3OH 93:7) afforded the ethyl ester 7 in a pure form (80 mg,
85%). 1H NMR (CDCl3, 300 MHz): d=8.35 (d, JHH =16.3 Hz, 2H), 8.17
(d, JHH =8.1 Hz, 2H), 7.57 (d, JHH =8.4 Hz, 4H), 7.43 (dd, 6H), 7.06 (d,
Compound 11
Saponification: Ethyl ester 8 (21.4 mg, 0.02 mmol) was dissolved in a mix-
ture of EtOH (1.5 mL) and deionized water (0.75 mL), and then aqueous
1.0m NaOH (0.33 mL, 0.33 mmol) was added. The resulting reaction mix-
ture was stirred at RT until the complete consumption of the staring ma-
terial was observed by RP-HPLC (system A) and MS (ESI). The reaction
was quenched by adding concentrated HCl (30 mL, 0.36 mmol) and puri-
fied by RP-HPLC (system B, 1 injection, tR =22.7–25.0 min). The prod-
uct-containing fractions were lyophilized to give the carboxylic acid inter-
mediate as a blue amorphous powder (10.4 mg, 50%). HPLC (system A):
tR =15.9 min (compared to tR =19.9 min for ethyl ester 8); MS (ESI, posi-
tive mode): m/z calcd for C50H53BF2N4O8S2: 950.33; found: 951.20
[M+H]+.
J
HH =16.3 Hz, 2H), 6.64 (s, 2H), 4.41 (q, JHH =7.4 Hz, 2H), 3.49 (s, 4H),
3.17 (s, 4H), 2.37(s, 12H), 2.17 (s, 12H), 1.42 (t, JHH =7.2 Hz, 3H),
ACHTUNGTRENNUNG
1.39 ppm (s, 6H); 13C NMR (CDCl3, 75 MHz): d=166.0, 151.9, 140.3,
138.0, 136.8, 133.4, 132.1, 131.5, 128.9, 127.2, 123.3, 122.0, 118.5, 90.7,
86.6, 85.4, 61.4, 48.9, 44.3, 29.7, 15.0, 14.3 ppm; MS (ESI, positive mode):
m/z (%): calcd for C56H61BN6O2: 859.5; found: 860.3 [M+H]+ (100); ele-
mental analysis calcd (%) for C56H61BN6O2·H2O: C 76.52, H 7.22, N 9.56;
found: C 76.22, H 6.89, N 9.37.
Compound 8: Distyryl-BODIPY 5 (62 mg, 0.084 mmol) was dissolved in
dry DMF (3 mL). 1,3-Propanesultone (103 mg, 0.85 mmol) was added
and the resulting reaction mixture was stirred at 608C overnight, until
the complete consumption of the starting material was observed by TLC
analysis (EtOH/H2O 8:2). Thereafter, the reaction mixture was centri-
fuged. The recovered precipitated product was purified by chromatogra-
phy on a silica gel column with the following eluant (EtOH/H2O 7:3) and
finally recrystallized with a mixture of methanol and EtOAc to afford the
bis(sulfobetain) 8 in a pure form (36 mg, 60%). 1H NMR (CD3OD/
CDCl3, 300 MHz): d=8.03 (d, JHH =8.2 Hz, 2H), 7.56 (d, JHH =16.3 Hz,
Conversion into N-hydroxysuccinimidyl ester: BODIPY carboxylic acid
(10.4 mg, 8.9 mmol, 1 equiv) was dissolved in dry NMP (410 mL). A solu-
tion of TSTU (507 mL) in dry NMP (35.6 mg, 13.3 equiv) and a 2.0m solu-
tion of DIEA (35 mL) in dry NMP (70 mmol, 8 equiv) were sequentially
added and the resulting mixture was protected from light and stirred at
RT overnight. The reaction was checked for completion by RP-HPLC
(system A). The resulting crude NHS ester was used without further pu-
rification. HPLC (system A): tR =17.1 min (compared to tR =15.9 min for
the free carboxylic acid).
4H), 7.44 (d, JHH =8.1 Hz, 4H), 7.36 (d, JHH =8.3 Hz, 4H), 7.29 (d, JHH
=
Coupling
reaction:
a-Sulfo-b-alaninyl-a-sulfo-b-alanine
(80 mg,
8.2 Hz, 2H), 7.08 (d, JHH =16.2 Hz, 4H), 6.52 (s, 2H), 4.31 (s, 4H), 4.27
(m, 2H), 3.05 (s, 12H), 2.75 (m, 4H), 2.09 (m, 4H), 1.27 ppm (m, 9H);
13C NMR (CD3OD/CDCl3, 75 MHz): d=166.4, 152.6, 142.7, 139.6, 137.7,
136.7, 133.7, 132.8, 131.8, 131.0, 129.3, 127.5, 123.9, 121.0, 117.9, 86.7,
85.5, 61.6, 61.4, 53.4, 51.9, 51.8, 18.3, 15.2, 14.3 ppm; MS (ESI, positive
mode): m/z (%): calcd for C52H57BF2N4O8S2: 977.4; found: 979.4
[M+H]+ (100); the compound was too hygroscopic for suitable elemental
analysis.
0.15 mmol) was dissolved in aqueous 0.25m NaHCO3 buffer (pH 8.2,
1.2 mL) and the resulting solution was cooled to 48C. The crude solution
of NHS ester in NMP (see above) was added dropwise to this stirred so-
lution and the resulting reaction mixture was stirred at RT. The reaction
was checked for completion by RP-HPLC (system C). Thereafter, the
mixture was diluted with aqueous 50 mm TEAB and purified by RP-
HPLC (system D). The product-containing fractions were lyophilized to
give the TEA salt of water-soluble BODIPY. Desalting by ion-exchange
chromatography followed by lyophilization afforded the acid form of 11
as a blue amorphous powder (0.75 mg, yield 6%, mixture of two racemic
diastereomers). HPLC (system C): tR =11.9 min, purity 96%; MS (ESI,
negative mode): m/z (%): calcd for C56H63BF2N6O16S4: 1252.3; found:
1251.0 [MꢁH]ꢁ (100); this compound was obtained in too small amounts
to be well-characterized by NMR spectroscopy.
Compound 9: Distyryl-BODIPY 6 (50 mg, 0.054 mmol) was dissolved in
dry C2H4Cl2 (3 mL). 1,3-Propanesultone (66 mg, 0.54 mmol) was added
and the resulting reaction mixture was stirred at 608C overnight, until
the complete consumption of the starting material was observed by TLC
analysis (EtOH/H2O 8:2). Thereafter, the reaction mixture was centri-
fuged. The recovered precipitated product was purified by chromatogra-
phy on a silica gel column by using the following eluent (EtOH/H2O 7:3)
and finally recrystallized with a mixture of methanol and EtOAc to
afford the bis(sulfobetain) 9 in a pure form (38 mg, 60%). 1H NMR
Compound 13: Ethyl ester 10 (60 mg, 0.04 mmol) was dissolved in a mix-
ture of EtOH (2 mL) and deionized water (0.5 mL), and then NaOH
(18 mg, 0.4 mmol) was added. The resulting reaction mixture was stirred
at RT for 10 h until the complete consumption of the starting ester was
observed by TLC analysis (EtOH/H2O 7:3). The reaction was quenched
by adding aqueous 2% HCl solution until pH ꢀ7 was obtained, and sub-
sequent addition of EtOAc led to precipitation of the crude product,
which was isolated by centrifugation. Purification by flash chromatogra-
phy on a RP-C18 silica gel column by using the following eluent (H2O/
(CD3OD/CDCl3, 300 MHz): d=8.24 (d,
JHH =16.4 Hz, 2H), 8.16 (d,
J
HH =8.1 Hz, 2H), 7.61 (d, JHH =8.2 Hz, 4H), 7.55 (d, JHH =8.2 Hz, 4H),
7.44 (d, JHH =8.3 Hz, 2H), 7.14 (d, JHH =16.2 Hz, 2H), 6.67 (s, 2H), 4.49
(s, 4H), 4.39 (q, JHH =7.3 Hz, 2H), 4.09 (s, 4H), 3.69 (m, 4H), 3.28 (m,
4H), 3.21 (s, 12H), 3.14 (m, 10H), 2.90 (m, 4H), 2.24 (m, 4H), 1.39 ppm
(m, 9H); 13C NMR (CD3OD/CDCl3, 75 MHz): d=166.2, 151.5, 140.8,
Chem. Eur. J. 2012, 00, 0 – 0
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R. Ziessel, A. Romieu et al.
1
THF 95:5) afforded 13 in a pure form (15 mg, 25%). H NMR (CD3OD/
Compound 16: Distyryl-BODIPY 15 (100 mg, 0.11 mmol) was dissolved
D2O, 200 MHz): d=8.26 (d, JHH =16.2 Hz, 2H), 8.20 (d, JHH =7.9 Hz,
2H), 7.98 (s, 8H), 7.61 (d, JHH =16.2 Hz, 2H), 7.41 (d, JHH =8.3 Hz, 2H),
6.99 (s, 2H), 4.67 (s, 4H), 4.37 (s, 4H), 3.80 (m, 4H), 3.51 (m, 4H), 3.46
(s, 12H), 3.04 (s, 16H), 2.72 (m, 4H), 2.39 (m, 4H), 1.54 ppm (s, 6H);
13C NMR (CD3OD/D2O, 50 MHz): d=153.2, 144.1, 142.3, 139.6, 138.0,
136.5, 135.0, 133.6, 131.8, 129.5, 123.1, 120.8, 93.0, 79.7, 64.6, 64.3, 57.1,
20.6, 15.6 ppm; HPLC (system C): tR =4.3 min, purity 97%; MS (ESI,
positive mode): m/z (%): calcd for C66H81BN6O14S4: 1319.5; found:
1321.3 [M+H]+ (100), 660.6 [(M+2H)/2]2+ (50); elemental analysis calcd
(%) for C66H81BN6O14S4·H2O: C 59.18, H 6.25, N 6.27; found: C 58.99,
H 5.92, N 5.99.
in a mixture of absolute EtOH (4 mL) and triethylamine (3 mL). [PdCl2-
AHCTUNTGREGUN(NN PPh3)2] (8 mg, 0.01 mmol) was added and the reaction mixture was kept
under a CO atmosphere for 5 h and heated at 608C. Thereafter, volatiles
were removed and the resulting residue was dissolved in CH2Cl2. The or-
ganic layer was washed with saturated aqueous NaHCO3, deionized
water, dried over anhydrous MgSO4, and then evaporated to dryness.
The resulting residue was purified by chromatography on an Al2O3
column by using a step gradient of CH3OH (0–1%) in CH2Cl2 as the
mobile phase to afford the ethyl ester 16 in a pure form (80 mg, 85%).
1H NMR (CDCl3, 300 MHz): d=8.34 (d, JHH =16.4 Hz, 2H), 8.16 (d,
J
HH =8.2 Hz, 2H), 7.58 (d, JHH =8.1 Hz, 4H), 7.43 (d, JHH =8.4 Hz, 2H),
7.30 (d, JHH =8.4 Hz, 4H), 7.14 (d, JHH =16.5 Hz, 2H), 6.62 (s, 2H), 4.41
(q, JHH =7.1 Hz, 2H), 3.43 (s, 4H), 3.18 (s, 4H), 2.24 (s, 12H), 2.16 (s,
12H), 1.41 (t, JHH =7.2 Hz, 3H), 1.38 ppm (s, 6H); 13C NMR (CDCl3,
75 MHz): d=166.3, 152.3, 140.6, 139.8, 137.8, 136.2, 134.2, 131.4, 130.3,
129.6, 127.5, 121.4, 118.5, 90.6, 64.3, 61.5, 53.6, 49.0, 45.6, 44.1, 15.2,
14.5 ppm; MS (ESI, positive mode): m/z (%): calcd for C52H61BN6O2:
811.5; found: 813.5 [M+H]+ (100); elemental analysis calcd (%) for
C52H61BN6O2: C 76.83, H 7.56, N 10.34; found: C 76.53, H 7.32, N 10.11.
4-[(Dimethylamino)methyl]benzaldehyde (B): N,N-Dimethyl-N-(4-iodo-
benzyl)amine (500 mg, 1.92 mmol), anhydrous sodium formate (156 mg,
2.30 mmol), and [PdCl2ACHTUNGTRENNUNG(PPh3)2] (69 mg, 0.096 mmol) were dissolved in
dry DMF (10 mL). The resulting reaction mixture was stirred at 1008C
for 4 h with bubbling CO gas. Thereafter, the reaction mixture was dilut-
ed with EtOAc, washed with deionized water (3ꢅ70 mL), and dried over
anhydrous MgSO4. After evaporation, the resulting residue was purified
by chromatography on an Al2O3 column by using CH2Cl2 as the eluent to
1
Compound 17: BODIPY 16 (80 mg, 0.10 mmol) was dissolved in dry
DMF (2 mL). 1,3-Propanesultone (240 mg, 1.97 mmol) was added and
the resulting reaction mixture was stirred at 608C for 12 h. Then, addition
of EtOAc led to precipitation of the crude product, which was recovered
by centrifugation. Purification by flash chromatography on a RP-C18
silica gel column by using the following eluent (H2O/THF 85:15) afford-
ed 17 in a pure form (82 mg, 63%). 1H NMR (CD3OD/D2O, 300 MHz):
afford B in a pure form (283 mg, 91%). H NMR (CDCl3, 200 MHz): d=
9.93 (s, 1H), 7.77 (d, JHH =8.0 Hz, 2H), 7.42 (d, JHH =8.0 Hz, 2H), 3.43
(s, 2H), 2.20 ppm (s, 6H); other spectroscopic data are identical to those
already reported in the literature.[33]
Compound 14: BODIPY 1 (450 mg, 0.70 mmol) and 4-[(dimethylamino)-
methyl]benzaldehyde B (227 mg, 1.39 mmol) were dissolved in dry tolu-
ene (5 mL) and piperidine (0.5 mL). The resulting reaction mixture was
heated at 1408C in a Dean–Stark apparatus for 2 h. Thereafter, the
Dean–Stark receiver was removed and the mixture was heated with
argon bubbling until the solvent was completely removed. The resulting
crude product was treated with saturated aqueous NaHCO3 and deio-
nized water, and finally extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4 and evaporated to dryness. The resulting
residue was purified by chromatography on an Al2O3 column by using
CH2Cl2 as the eluent to afford the distyryl-BODIPY 14 (210 mg, 40%).
1H NMR (CDCl3, 300 MHz): d=7.84 (d, JHH =8.3 Hz, 2H), 7.70 (d,
d=8.31 (d, JHH =7.2 Hz, 2H), 8.28 (d, JHH =16.1 Hz, 2H), 7.93 (d, JHH
=
7.3 Hz, 4H), 7.84 (d, JHH =7.3 Hz, 4H), 7.61 (d, JHH =16.4 Hz, 2H), 7.05
(s, 2H), 4.69 (s, 4H), 4.50 (q, JHH =6.9 Hz, 2H), 4.38 (s, 4H), 3.62 (m,
4H), 3.54 (m, 4H), 3.21 (s, 12H), 3.07 (s, 12H), 3.02 (m, 4H), 2.64 (m,
4H), 2.41 (m, 4H), 2.15 (m, 4H), 1.56 (s, 6H), 1.50 ppm (t, JHH =6.6 Hz,
3H); 13C NMR (CD3OD/D2O, 75 MHz): d=167.9, 153.2, 143.7, 140.8,
136.3, 135.6, 132.9, 131.7, 130.3, 129.8, 122.3, 120.6, 85.4, 69.4, 63.8, 56.8,
51.2, 50.0, 49.7, 20.1, 15.3, 14.8 ppm; MS (ESI, positive mode): m/z (%):
calcd for C64H85BN6O14S4: 1299.5; found: 1301.3 [M+H]+ (100), 651.2
[(M+2H)/2]2+
(55);
elemental
analysis
calcd
(%)
for
J
HH =16.6 Hz, 2H), 7.57 (d, JHH =8.3 Hz, 4H), 7.33 (d, JHH =8.2 Hz, 4H),
C64H85BN6O14S4·1.5H2O: C 57.86, H 6.68, N 6.33; found: C 57.52, H 6.53,
N 6.69.
7.24 (d, JHH =16.4 Hz, 2H), 7.08 (d, JHH =8.2 Hz, 2H), 6.63 (s, 2H), 3.49
(s, 4H), 2.27 (s, 12H), 1.47 ppm (s, 6H); 13C NMR (CDCl3, 75 MHz): d=
142.1, 138.5, 136.5, 135.8, 134.9, 130.7, 127.8, 119.2, 118.3, 64.0, 45.3, 29.9,
22.9, 15.2, 14.3 ppm; MS (ESI, positive mode): m/z (%): calcd for
C39H40BF2IN4: 739.2; found: 740.2 [M+H]+ (100); elemental analysis
calcd (%) for C39H40BF2IN4: C 63.26, H 5.44, N 7.57; found: C 63.03,
H 5.13, N 7.34.
Compound 18: Ethyl ester 17 (70 mg, 0.05 mmol) was dissolved in a mix-
ture of EtOH (2 mL) and deionized water (0.5 mL), and then NaOH
(21 mg, 0.5 mmol) was added. The resulting reaction mixture was stirred
at RT for 10 h until complete consumption of the starting ester was ob-
served by TLC analysis (EtOH/H2O 7:3). The reaction was quenched by
adding aqueous 2% HCl solution until pH ꢀ7 was obtained, and subse-
quent addition of EtOAc led to precipitation of the crude product, which
was isolated by centrifugation. Purification by flash chromatography on
a RP-C18 silica gel column by using a step gradient of THF (5–10%) in
deionized water as the mobile phase afforded 18 in a pure form (60 mg,
Compound 15:
A solution of 1-dimethylamino-2-propyne (121 mg,
1.46 mmol) in dry THF (3 mL) was placed in a Schlenk tube under an
argon atmosphere. A 1.0m solution of EtMgBr in THF (1.28 mL) was
added and the reaction mixture was stirred at 608C for 1 h. The resulting
anion was then transferred through a cannula to a solution of distryryl-
BODIPY 14 (260 mg, 0.35 mmol) in dry THF (3 mL) previously placed
in a Schlenk tube under an argon atmosphere. The resulting reaction
mixture was stirred at 608C for 15 min until the complete consumption
of the starting BODIPY was observed by TLC analysis. Then, the mix-
ture was sequentially treated with deionized water and brine and then ex-
tracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4
and evaporated to dryness. The crude product was purified by chroma-
tography on an Al2O3 column with a step gradient of CH3OH (0–1%) in
88%). 1H NMR (CD3OD/D2O, 300 MHz): d=8.26 (d,
2H), 8.21 (d, JHH =8.3 Hz, 2H), 7.92 (d, JHH =8.2 Hz, 4H), 7.83 (d, JHH
JHH =16.3 Hz,
=
8.2 Hz, 4H), 7.58 (d, JHH =16.2 Hz, 2H), 7.44 (d, JHH =8.3 Hz, 2H), 7.01
(s, 2H), 4.66 (s, 4H), 4.36 (s, 4H), 3.64 (m, 4H), 3.51 (m, 4H), 3.21 (s,
12H), 3.06 (s, 12H), 3.03 (m, 4H), 2.65 (t, JHH =6.8 Hz, 4H), 2.42 (m,
4H), 2.14 (m, 4H), 1.53 ppm (s, 6H); 13C NMR (CD3OD/D2O, 75 MHz):
d=152.9, 143.8, 139.9, 135.5, 133.0, 131.6, 129.7, 122.2, 120.5, 85.3, 80.2,
69.3, 67.1, 63.7, 56.8, 51.1, 20.0, 15.3 ppm; MS (ESI, positive mode): m/z
(%): calcd for C62H81BN6O14S4: 1271.5; found: 1273.4 [M+H]+ (100),
637.2 [(M+2H)/2]2+ (35); elemental analysis calcd (%) for
C62H81BN6O14S4·2.5H2O: C 56.48, H 6.57, N 6.37; found: C 56.18, H 6.29,
N 6.76.
CH2Cl2 as the mobile phase to give the bis
(247 mg, 81%). 1H NMR (CDCl3, 300 MHz): d=8.34 (d, JHH =16.3 Hz,
2H), 7.80 (d, JHH =8.3 Hz, 2H), 7.57 (d, JHH =8.0 Hz, 4H), 7.29 (d, JHH
ACHTUNGTREN(NUGN ethynyl) BODIPY 15
=
8.3 Hz, 4H), 7.13 (d, JHH =16.4 Hz, 2H), 7.06 (d, JHH =8.4 Hz, 2H), 6.61
(s, 2H), 3.42 (s, 4H), 3.17 (s, 4H), 2.23 (s, 12H), 2.15 (s, 12H), 1.43 ppm
(s, 6H); 13C NMR (CDCl3, 75 MHz): d=152.2, 140.2, 139.8, 138.3, 137.6,
136.2, 135.4, 134.1, 131.6, 130.8, 129.6, 127.5, 121.4, 118.4, 94.7, 90.7, 64.3,
49.0, 45.5, 44.3, 15.3 ppm; MS (ESI, positive mode): m/z (%): calcd for
C49H56BIN6: 865.4; found: 866.2 [M+H]+ (100); elemental analysis calcd
(%) for C49H56BIN6·2H2O: C 65.19, H 6.70, N 9.31; found: C 64.84,
H 6.62, N 9.04.
Preparation of BODIPY/cyanine–protein conjugates
General procedure for the conversion into N-hydroxysulfosuccinimidyl
ester: The water-soluble BODIPY dye carboxylic acid (0.37 mmol, weigh-
ed in a 0.5 mL Eppendorf microtutube) was dissolved in deionized water
(50 mL). An aqueous solution of water-soluble carbodiimide (30 mL,
EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride,
12–15 equiv) and an aqueous solution of sulfo-NHS (10 mL, 0.17 mg,
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Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
0.78 mmol, 2 equiv) were sequentially added and the resulting reaction
mixture was protected from light and periodically stirred by use of
a vortex mixer. The reaction was checked for completion by RP-HPLC
(system A or C). The resulting N-hydroxysulfosuccinimidyl ester was
used in the next labeling step without purification.
[4] For selected examples, see: a) L.-Q. Ying, B. Branchaud, Bioconju-
gate Chem. 2011, 22, 865–869; b) F. Shao, H. Yuan, L. Josephson, R.
Weissleder, S. A. Hilderbrand, Dyes Pigm. 2011, 90, 119–122; c) F.
Shao, R. Weissleder, S. A. Hilderbrand, Bioconjugate Chem. 2008,
19, 2487–2491; d) W. Pham, L. Cassell, A. Gillman, D. Koktysh,
J. C. Gore, Chem. Commun. 2008, 1895–1897; e) H. Lee, J. C.
Mason, S. Achilefu, J. Org. Chem. 2008, 73, 723–725; f) M. V. Red-
dington, Bioconjugate Chem. 2007, 18, 2178–2190; g) C. Bouteiller,
G. Clavꢀ, A. Bernardin, B. Chipon, M. Massonneau, P.-Y. Renard,
A. Romieu, Bioconjugate Chem. 2007, 18, 1303–1317; h) H. Lee,
J. C. Mason, S. Achilefu, J. Org. Chem. 2006, 71, 7862–7865; i) S. R.
Mujumdar, R. B. Mujumdar, C. M. Grant, A. S. Waggoner, Biocon-
jugate Chem. 1996, 7, 356–362.
Sulfo-NHS ester from 12: HPLC (system C): tR =10.3 min (compared to
tR =9.7 min for water-soluble BODIPY carboxylic acid 12).
Sulfo-NHS ester from 13: HPLC (system A): tR =12.1 min (compared to
tR =13.1 min for water-soluble BODIPY carboxylic acid 13).
Fluorescent labeling of proteins: The solution of N-hydroxysulfosuccini-
midyl ester (see above, 45 mL, 185 nmol, 14- or 31-fold excess according
to protein) was added to
a ,
solution of BSA (500 mL, 1.8 mgmLꢁ1
13 nmol) or anti-HA mAb (1.8 mgmLꢁ1, 6 nmol) in phosphate buffer
(pH 7.4). The resulting mixture was protected from the light and periodi-
cally stirred by use of a vortex mixer. The reaction was left at 48C over-
night. Thereafter, the BODIPY–BSA or BODIPY–mAb conjugate was
purified by size-exclusion chromatography with an Econo-Pac Disposable
chromatography column (Bio-Rad, #732–1010) filled with an aqueous so-
lution of Sephadex G-25 Fine (Amersham Biosciences AB, 15ꢅ40 mm
bed), equilibrated with PBS (0.01m phosphate, 0.015m NaCl, pH 7.5).
The dye-to-protein ratio (F/P) of these conjugates was determined spec-
trophotometrically by measuring their absorbance at 280 and 642 nm and
inserting the measured values into Equation (2) in which A280 is the ab-
[5] R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis, A. S.
Waggoner, Bioconjugate Chem. 1993, 4, 105–111.
[6] For selected examples, see: a) A. Romieu, D. Tavernier-Lohr, S.
Pellet-Rostaing, M. Lemaire, P.-Y. Renard, Tetrahedron Lett. 2010,
51, 3304–3308; b) G. Y. Mitronova, V. N. Belov, M. L. Bossi, C. A.
Wurm, L. Meyer, R. Medda, G. Moneron, S. Bretschneider, C. Egg-
eling, S. Jakobs, S. W. Hell, Chem. Eur. J. 2010, 16, 4477–4488; c) K.
Kolmakov, V. N. Belov, C. Ringenmann, V. Mꢇller, C. Eggeling,
S. W. Hell, Chem. Eur. J. 2010, 16, 158–166; d) K. Kolmakov, V. N.
Belov, C. A. Wurm, B. Harke, M. Leutenegger, C. Eggeling, S. W.
Hell, Eur. J. Org. Chem. 2010, 3593–3610; e) V. P. Boyarskiy, V. N.
Belov, R. Medda, B. Hein, M. Bossi, S. W. Hell, Chem. Eur. J. 2008,
14, 1784–1792.
[7] Some water-soluble BODIPY dyes have been recently reported but
none of them exhibit spectral properties in the red region, see: a) S.
Zhu, J. Zhang, G. Vegesna, F.-T. Luo, S. A. Green, H. Liu, Org. Lett.
2011, 13, 438–441; b) M. Brellier, G. Duportail, R. Baati, Tetrahe-
dron Lett. 2010, 51, 1269–1272; c) L. Jiao, J. Li, S. Zhang, C. Wei, E.
Hao, M. G. H. Vicente, New J. Chem. 2009, 33, 1888–1893; d) O.
Dilek, S. L. Bane, Bioorg. Med. Chem. Lett. 2009, 19, 6911–6913;
e) L. Li, B. Nguyen, K. Burgess, Bioorg. Med. Chem. Lett. 2008, 18,
3112–3116; f) L. Li, J. Han, B. Nguyen, K. Burgess, J. Org. Chem.
2008, 73, 1963–1970; g) C. Thivierge, R. Bandichhor, K. Burgess,
Org. Lett. 2007, 9, 2135–2138; h) N. J. Meltola, R. Wahlroos, A. E.
Soini, J. Fluoresc. 2004, 14, 635–647.
P
sorbance of the protein at 280 nm, e280 is the extinction coefficient of the
protein at 280 nm, Amax is the absorbance of the BODIPY label as its ab-
F
sorption maximum, emax is the extinction coefficient of the fluorophore
F
at the absorption maximum, and e280 is the extinction coefficient of the
fluorophore at 280 nm (10767 for 12, 12618 for 13, and 25464mꢁ1 cmꢁ1
for Cy 5.0). We used a correction procedure for the visible part of the
spectra at high F/P ratios. Specifically, we considered the integrated area
under the visible part of the spectrum (and not the height of the dye
peak) for quantization of the dye in solution. We assumed that the quan-
tity of the dye is proportional to the integrated visible absorption spec-
trum or area under the curve (AUC) from 475 to 750 nm, and calculated
a correction factor for each conjugate equal to the ratio of the AUC for
the conjugate to the AUC for the free nonbound label (normalized spec-
tra). Anti-HA mAb and BSA have an extinction coefficient at 280 nm of
295000 and 43824mꢁ1 cmꢁ1 respectively.
[8] S. Atilgan, Z. Ekmekci, A. L. Dogan, D. Guc, E. U. Akkaya, Chem.
Commun. 2006, 4398–4400.
[9] S. Atilgan, T. Ozdemir, E. U. Akkaya, Org. Lett. 2008, 10, 4065–
4067.
P
F
F
ð2Þ
F=P ¼ Amax e280=ðA280 emax þ Amax e280
Þ
[10] a) M. Tasior, D. F. OꢃShea, Bioconjugate Chem. 2010, 21, 1130–
1133; b) M. Tasior, J. Murtagh, D. O. Frimannsson, S. O. McDonnell,
D. F. OꢃShea, Org. Biomol. Chem. 2010, 8, 522–525.
Further fluorescent labeling experiments were performed with sulfoindo-
cyanine dye Cy 5.0 (Femax =250000mꢁ1 cmꢁ1) under the same conditions.
[11] A. Romieu, T. Bruckdorfer, G. Clave, V. Grandclaude, C. Massif, P.-
Y. Renard, Org. Biomol. Chem. 2011, 9, 5337–5342.
[12] A. Romieu, D. Brossard, M. Hamon, H. Outaabout, C. Portal, P.-Y.
Renard, Bioconjugate Chem. 2008, 19, 279–289.
Acknowledgements
[13] S.-L. Niu, G. Ulrich, P. Retailleau, J. Harrowfield, R. Ziessel, Tetra-
hedron Lett. 2009, 50, 3840–3844.
[14] S. L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P.-Y. Renard, A. Romieu,
Org. Lett. 2009, 11, 2049–2052.
[15] S. L. Niu, C. Massif, G. Ulrich, R. Ziessel, P.-Y. Renard, A. Romieu,
Org. Biomol. Chem. 2011, 9, 66–69.
[16] T. N. Singh-Rachford, A. Haefele, R. Ziessel, F. N. Castellano, J.
Am. Chem. Soc. 2008, 130, 16164–16165.
This work was supported by the Agence Nationale de la Recherche (Pro-
gramme Blanc 2009, ANR-09-BLAN-0081—01), especially by a PhD
grant to C. Massif, La Rꢀgion Haute-Normandie through the CRUNCh
program (CPER 2007—2013), and the Institut Universitaire de France
(IUF). We thank Dr. G. Clavꢀ for the synthesis of sulfoindocyanine dye
Cy 5.0 and Dr. H. Volland (iBiTecS, LERI, CEA-Saclay) for the kind gift
of anti-HA mAb clone 12A5. We warmly thank Professor J. Harrowfield
from ISIS in Strasbourg for commenting on this manuscript before publi-
cation.
[17] a) A. Schoenberg, I. Bartoletti, R. F. Heck, J. Org. Chem. 1974, 39,
3318–3326; b) A. El-ghayoury, R. Ziessel, Tetrahedron Lett. 1998,
39, 4473–4476.
[18] R. Ziessel, P. Retailleau, K. J. Elliott, A. Harriman, Chem. Eur. J.
2009, 15, 10369–10374.
[1] S. A. Hilderbrand, R. Weissleder, Curr. Opin. Chem. Biol. 2010, 14,
71–79.
[19] W. Bannwarth, R. Knorr, Tetrahedron Lett. 1991, 32, 1157–1160.
[20] D. Tleugabulova, Z. Zhang, J. D. Brennan, J. Phys. Chem. B 2002,
106, 13133–13138.
[21] a) J. Pauli, M. Grabolle, R. Brehm, M. Spieles, F. M. Hamann, M.
Wenzel, I. Hilger, U. Resch-Genger, Bioconjugate Chem. 2011, 22,
1298–1308, and references therein; b) E. G. Matveeva, E. A. Ter-
[2] For selected reviews, see: a) J. O. Escobedo, O. Rusin, S. Lim, R. M.
Strongin, Curr. Opin. Chem. Biol. 2010, 14, 64–70; b) L. D. Lavis,
R. T. Raines, ACS Chem. Biol. 2008, 3, 142–155; c) A. Gꢆmez-
Hens, M. P. Aguilar-Caballos, TrAC Trends Anal. Chem. 2004, 23,
127–136.
[3] M. S. T. GonÅalves, Chem. Rev. 2009, 109, 190–212.
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
13
&
ÞÞ
These are not the final page numbers!

R. Ziessel, A. Romieu et al.
petschnig, M. Stevens, L. Patsenker, O. S. Kolosova, Z. Gryczynski,
I. Gryczynski, Dyes Pigm. 2009, 80, 41–46.
[28] A. A. Wilson, R. F. Dannals, H. T. Ravert, J. J. Frost, H. N. Wagner,
J. Med. Chem. 1989, 32, 1057–1062.
[22] P. Didier, G. Ulrich, Y. Mely, R. Ziessel, Org. Biomol. Chem. 2009,
7, 3639–3642.
[29] R. E. Counsell, T. T.-J. Yu (University of Michigan, MI), US-A
4048297, 1977.
[23] W. Qin, T. Rohand, W. Dehaen, J. N. Clifford, K. Driesen, D. Bel-
jonne, B. Van Averbeke, M. Van der Auweraer, N. Boens, J. Phys.
Chem. A 2007, 111, 8588–8597.
[24] K. Rurack, M. Kollmannsberger, J. Daub, New J. Chem. 2001, 25,
289–292.
[25] a) C. Chidley, K. Mosiewicz, K. Johnsson, Bioconjugate Chem. 2008,
19, 1753–1756; b) D. Maurel, J. Kniazeff, G. Mathis, E. Trinquet, J.-
P. Pin, H. Ansanay, Anal. Biochem. 2004, 329, 253–262.
[26] a) J. D. Debord, L. A. Lyon, Bioconjugate Chem. 2007, 18, 601–604;
b) Y. Gao, I. Kyratzis, Bioconjugate Chem. 2008, 19, 1945–1950.
[27] For recent reviews, see: a) A. Razgulin, N. Ma, J. Rao, Chem. Soc.
Rev. 2011, 40, 4186–4216; b) C. R. Drake, D. C. Miller, E. F. Jones,
Curr. Org. Synth. 2011, 8, 498–520.
[30] N. Miyaura, A. Suzuki, Org. Synth. 1990, 68, 130.
[31] D. R. Coulson, L. C. Satek, S. O. Grim in Inorganic Syntheses,
Vol. 13 (Ed.: F. A. Cotton), McGraw-Hill, New York, 1972, pp. 121–
124.
[32] J. Olmsted III, J. Phys. Chem. 1979, 83, 2581–2584.
[33] a) R. Sheng, Y. Xu, C. Hu, J. Zhang, X. Lin, J. Li, B. Yang, Q. He,
Y. Hu, Eur. J. Med. Chem. 2009, 44, 7–17; b) T. Horiuchi, M.
Nagata, M. Kitagawa, K. Akahane, K. Uoto, Bioorg. Med. Chem.
2009, 17, 7850–7860.
Received: November 17, 2011
Published online: && &&, 0000
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Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Water-Soluble Red-Emitting Borondipyrromethene (BODIPY) Dyes
FULL PAPER
Go bio! Water-soluble red-emitting
borondipyrromethene (BODIPY)
derivatives have been prepared by
using mild sulfonation procedures (see
figure). The sterically demanding sub-
stituents on boron and sulfonate
groups on the top/bottom parts of the
distyryl-BODIPY scaffold effectively
suppress their aggregation in aqueous
solution. These bright fluorophores
show promise as biomolecule labels.
Biolabeling
S.-l. Niu, C. Massif, G. Ulrich,
P.-Y. Renard, A. Romieu,*
R. Ziessel* ..................... &&&& — &&&&
Water-Soluble Red-Emitting Distyryl-
Borondipyrromethene (BODIPY)
Dyes for Biolabeling
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
15
&
ÞÞ
These are not the final page numbers!