Water-soluble BODIPY derivatives

Article abstract of DOI:10.1021/ol900302n

New, water-soluble BODIPY dyes have been readily obtained from various BODIPY cores by reactions involving the introduction of novel sulfonated peptide chains by either coupling or substitution to give dimethylpropargylamine derivatives subsequently quate

Full text of DOI:10.1021/ol900302n

ORGANIC
LETTERS
2009
Vol. 11, No. 10
2049-2052
Water-Soluble BODIPY Derivatives
Song Lin Niu,† Gilles Ulrich,† Raymond Ziessel,*,† Agneta Kiss,‡
Pierre-Yves Renard,‡,§,¶ and Anthony Romieu*,‡,§
Laboratoire de Chimie Organique et Spectroscopies AVance´es (LCOSA), CNRS,
UniVersite´ Louis Pasteur, Ecole de Chimie, Polyme`res, Mate´riaux de Strasbourg
(ECPM), 25 rue Becquerel, 67087 Strasbourg, Cedex 02, France, Equipe de Chimie
Bio-Organique, UniVersite´ de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan,
France, IRCOF, COBRA-CNRS UMR 6014 & FR 3038, rue Lucien Tesnie`re,
76131 Mont-Saint-Aignan, France, and Institut UniVersitaire de France,
103, bd Saint-Michel, 75005 Paris, France
ziessel@chimie.u-strasbg.fr; anthony.romieu@uniV-rouen.fr
Received February 12, 2009
ABSTRACT
New, water-soluble BODIPY dyes have been readily obtained from various BODIPY cores by reactions involving the introduction of novel
sulfonated peptide chains by either coupling or substitution to give dimethylpropargylamine derivatives subsequently quaternized by reaction
with propanesultone.
Fluorescent organic dyes are widely used as nonradioactive
labels in biological analysis1 or as biomarkers in biomedical
applications including imaging diagnosis.2 Indeed, the attributes
of high sensitivity and high spatiotemporal resolution and the
availability of simple and rapid analytical systems make
fluorescence techniques powerful tools for the interrogation of
small sample volumes, small numbers of analyte molecules,
and even the study of single components in complex biological
systems. However, among the myriad of available synthetic
fluorescent molecules exhibiting high extinction coefficients,
high quantum yields, narrow emission bands, and photostabil-
ity,3 many have limited utility due to poor solubility. For most
biological applications both good water solubility and resistance
to the formation of nonfluorescent dimer and higher aggregates
(especially after conjugation to biological material) are essential.
Generally, solubility has been imported by either introducing
ionizable hydrophilic groups (carboxylic acid, phosphonic acid,
sulfonic acid, and ammonium groups) within the core structure
of a fluorescent dye4 or by grafting the hydrophobic fluorophore
to hydrophilic (bio)polymers (carbohydrates, oligonucleotides
and polyethylene glycol).5 Both strategies have been extensively
used with benzo[a]phenoxazine, xanthene and cyanine dyes,
resulting in the development of several families of novel water-
soluble fluorescent labels.6 Some of them are commercially
(4) For recent examples, see: (a) Peneva, K.; Mihov, G.; Nolde, F.;
Rocha, S.; Hotta, J.-i.; Braeckmans, K.; Hofkens, J.; Uji-i, H.; Herrmann,
A.; Mu¨llen, K. Angew. Chem., Int. Ed. 2008, 47, 3372–3375. (b) Reddington,
M. V. Bioconjugate Chem. 2007, 18, 2178–2190. (c) Wang, H.; Lu, Z.;
Lord, S. J.; Moerner, W. E.; Twieg, R. J. Tetrahedron Lett. 2007, 48, 3471–
3474.
† LCOSA.
‡ COBRA.
§ Universite´ de Rouen.
¶ Institut Universitaire de France.
(1) Haugland, R. P.; Spence, M. T. Z.; Johnson, I. D.; Basey, A. The
Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 10th
ed.; Molecular Probes: Eugene, OR, 2005.
(2) Ballou, B.; Ernst, L. A.; Waggoner, A. S. Curr. Med. Chem. 2005,
12, 795–805.
(5) For selected examples, see: (a) Atilgan, S.; Ozdemir, T.; Akkaya,
E. U. Org. Lett. 2008, 10, 4065–4067. (b) Goussu, C.; Vasseur, J.-J.; Bazin,
H.; Trinquet, E.; Maurin, F.; Morvan, F. Bioconjugate Chem. 2005, 16,
465–470. (c) Katritzky, A. R.; Cusido, J.; Narindoshvili, T. Bioconjugate
Chem. 2008, 19, 1471–1475. (d) Reddington, M. V. J. Chem. Soc., Perkin
Trans. 1998, 1, 143–148.
(3) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155.
10.1021/ol900302n CCC: $40.75
Published on Web 04/20/2009
 2009 American Chemical Society

available (e.g., Alexa Fluor,7 Cy dyes,8 etc.) and widely used
as fluorescent tags in biotechnological applications. Usually,
new synthetic routes are required to introduce the desired water-
solubilizing groups, which have to be masked during the
synthesis to prevent cross reactivity and/or troublesome puri-
fications. It is thus of prime interest to be able to modify
postsynthetically the fluorophore skeleton with a flexible and
tunable hydrophilic moiety, as we have previously illustrated
with cyanine- and rhodamine-based fluorescent species.9 Sur-
prisingly, little synthetic effort has been devoted to the water-
solubilization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BO-
DIPY) dyes and few hydrophilic labeling reagents derived from
this fluorescent core have been reported.10 Recently, Burgess
et al. explored three different strategies to introduce one or two
sulfonate groups onto the hydrophobic BODIPY core, either
in the 2- and 6-positions through Heck-type coupling11 or
electrophilic sulfonation with chlorosulfonic acid12 inspired by
the work of Boyer et al.13 or in the 3-position by SNAr reaction
of a chloro substituent with 2-mercaptosulfonic acid.14 Only
the synthetic approach based on the electrophilic substitution
reaction gave water-soluble BODIPY derivatives in good yields
without requiring a prior functionalization of the pyrrole
moieties (e.g., with chloro substituents). This solubilization
strategy could be applied to BODIPY dyes to get the corre-
sponding mono- or disulfonated derivatives, but the water-
solubilizing moiety cannot be finely tuned. Thus, it is useful to
explore alternative methods, allowing the introduction of a large
number of sulfonate groups in a single step by means of an
easy-to-handle reagent, especially for an efficient enhancement
of water solubility of fluorescent BODIPY cores bearing
additional aromatic rings in the meso, 3- and 5-positions (i.e.,
styryl-BODIPY derivatives).
of the widely used rhodamine 6G (R6G) and sulfoindocyanine
dye Cy 5.0. As their dipyrromethene unit contains a m-phenyl
substituent carrying a carboxylic acid functional group, a peptide
coupling reaction with a hydrophilic linker bearing both an
amino and several sulfonate groups was anticipated to be a direct
way to introduce water-solubilizing residues. To reach the target,
the acids 1 and 8 were prepared in two steps from the
corresponding phenyliodo derivatives using first a carboalkoxy-
lation reaction promoted by [Pd(PPh3)2Cl2] with ethanol as the
nucleophile, followed by a saponification reaction with KOH
in a mixture of methanol and water (for the synthesis details,
see Supporting Information). The carboxylic acid 1 was first
converted quantitatively into the corresponding N-hydroxysuc-
cinimidyl (NHS) ester by treatment with N,N,N′,N′-tetramethyl-
O-(N-succinimidyl)uronium tetrafluoroborate (TSTU)/diisopro-
pylethylamine (DIEA) in dry N-methylpyrrolidone (NMP)
(Scheme 1).
Scheme 1.
Synthesis of Water-Soluble BODIPY 3a
a Compound 3 was obtained as a triethylammonium salt after reverse-
phase (RP)-HPLC purification with aqueous triethylammonium bicarbonate
(TEAB) buffer as mobile phase.
Thereafter, acylation of the primary amino group of 2 with
the in situ prepared active ester in a mixture of bicarbonate
buffer (pH 8.5) and NMP gave the desired water-soluble
analogue 3 in a moderate yield (38% after RP-HPLC purifica-
tion). The use of NMP as organic cosolvent was not sufficient
to completely prevent precipitation of the intermediate NHS
ester, which slowly reacts with 2 and is prone to hydrolysis to
give back the starting BODIPY 1 (which was recovered
unmodified in 30% yield). The same postsynthetic sulfonation
procedure was applied to the styryl-BODIPY 8, but its greater
hydrophobic character could not be countered by the introduc-
tion of only two sulfonate groups. Thus, we explored the
chemistry of the tripeptide (R-sulfo-ꢀ-alanine)3 7, which was
readily synthesized in two steps from the N-Fmoc dipeptide 4
used in the preparation of disulfonated linker 2 (Scheme 2).
The diethylammonium salt of this highly hydrophilic linker was
isolated in good yield by conventional liquid-liquid extraction,
and its structure was confirmed by detailed measurements,
including ESI mass spectrometry and NMR analyses.
To avoid complete precipitation of the active ester in the
NMP-water mixture (observed in our first attempt for deriva-
tization of 8 with 7), which would prevent an efficient acylation,
the previous synthetic protocol was modified by adding a
transesterification step with N-hydroxysulfosuccinimide (sulfo-
NHS). The resulting sulfo-NHS ester 9 was found to be
conveniently soluble in the reaction mixture, and its derivati-
zation with 7 afforded the target water-soluble styryl-BODIPY
Herein, we report two straightforward methods to introduce
(poly)sulfonated linkers derived from R-sulfo-ꢀ-alanine or
sulfobetaine onto BODIPY scaffolds by postsynthetic deriva-
tization through amide formation, alkyne-coupling, and B-F
substitution reactions. The spectral properties of the resulting
water-soluble BODIPY derivatives were then evaluated under
physiological conditions.
First, we focused on the water solubilization of two different
BODIPYs, 1 and 8, whose spectral properties are close to those
(6) For recent examples, see: (a) Boyarskiy, V. P.; Belov, V. N.; Medda,
R.; Hein, B.; Bossi, M.; Hell, S. W. Chem.sEur. J. 2008, 14, 1784–1792.
(b) Ho, N.-H.; Weissleder, R.; Tung, C.-H. Tetrahedron 2006, 62, 578–
585. (c) Jose, J.; Burgess, K. J. Org. Chem. 2006, 71, 7835–7839.
(7) Berlier, J. E.; Rothe, A.; Buller, G.; Bradford, J.; Gray, D. R.;
Filanoski, B. J.; Telford, W. G.; Yue, S.; Liu, J.; Cheung, C.-Y.; Chang,
W.; Hirsch, J. D.; Beechem, J. M.; Haugland, R. P.; Haugland, R. P.
J. Histochem. Cytochem. 2003, 51, 1699–1712.
(8) (a) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.;
Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105–111. (b) Mujumdar,
S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S. Bioconjugate Chem.
1996, 7, 356–362.
(9) Romieu, A.; Brossard, D.; Hamon, M.; Outaabout, H.; Portal, C.;
Renard, P.-Y. Bioconjugate Chem. 2008, 19, 279–289.
(10) Meltola, N. J.; Wahlroos, R.; Soini, A. E. J. Fluorescence 2004,
14, 635–647.
(11) Thivierge, C.; Bandichhor, R.; Burgess, K. Org. Lett. 2007, 9, 2135–
2138.
(12) Li, L.; Han, J.; Nguyen, B.; Burgess, K. J. Org. Chem. 2008, 73,
1963–1970.
(13) Boyer, J. H.; Haag, A. M.; Sathyamoorthi, G.; Soong, M. L.;
Thangaraj, K.; Pavlopoulos, T. G. Heteroat. Chem. 1993, 4, 39–49.
(14) Li, L.; Nguyen, B.; Burgess, K. Bioorg. Med. Chem. Lett. 2008,
18, 3112–3116.
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Org. Lett., Vol. 11, No. 10, 2009

Scheme 2
.
Synthesis of Trisulfonated Peptidyl Linker 7
Scheme 4
.
Synthesis of Water-Soluble BODIPYs 13 and 16
using Sultone Alkylation
Scheme 3.
Synthesis of Water-Soluble Styryl-BODIPY 10a
Pd(II) and Cu(I) and yielded compound 12 in 42% yield.
Subsequent alkylation with 1,3-propanesultone (in excess) in
dry toluene afforded the betaine 13, which precipitated during
the course of the reaction and could be isolated by centrifugation
without additional treatment (58% isolated yield). Substitution
of the fluoro groups proceeded readily with the Grignard reagent
of 1-dimethylamino-2-propyne, providing compound 15 in 63%
yield. Again, alkylation with 1,3-propanesultone provided the
betaine 16 in 62% isolated yield. Dyes 13 and 16 were soluble
in water and related aqueous buffers in the concentration range
of 0.3-0.75 mM.
The photophysical properties of the novel water-soluble
BODIPY dyes were evaluated under simulated physiological
conditions [i.e., phosphate-buffered saline (PBS), pH 7.3] and
are collected in Table 1. As would be expected from previous
studies, the presence of a hydrophilic linker on the 8-phenyl
substituent or sulfobetaine fragments at boron does not affect
the spectral properties of the dyes (Figure 1 and Table 1).
However, in the case of the 2,6-disubstitution in dye 13, a
bathochromic shift of 9 nm is observed in the emission spectra
with respect to dye 16 when measured under the same
conditions (Figure 2, top). In the case of dye 16 the absorption
spectra in PBS exhibits two strong peaks at 522 and 567 nm.
The latter is characteristic of an aggregate and disappears in
ethanol (single absorption at 522 nm, Figure 2, top). In PBS or
ethanol, a single emission is observed at 532 nm with quantum
a Compound 10 was obtained as a triethylammonium salt when RP-HPLC
purification was performed with aqueous TEAB buffer as the mobile phase.
10 (41% after RP-HPLC purification, Scheme 3). All spectro-
scopic data, especially NMR and mass spectra, were in
agreement with the structures assigned for 3 and 10. As
expected, these triethylammonium salts were found to be
perfectly soluble in water and related aqueous buffers in the
concentration range (1 µM to 10 mM) suitable for biomolecular
labeling applications.
To explore further the synthetic opportunities offered by
alkynylation protocols for the 2,6-position of a BODIPY
framework15 and by recently developed boron chemistry,16 we
designed and prepared additional water-soluble derivatives using
a novel strategy (Scheme 4).
The idea was to use 1-dimethylamino-2-propyne as the
module to carry the nucleophile and to quaternarize the
dimethylamino group with 1,3-propanesultone to provide a
zwitteranionic fragment (betaine). The efficient solvation of the
zwitterionic unit proved to be sufficient to give overall solubility.
The use of sultone alkylation is commonly used in medicinal
chemistry to convert easily (cyclic) aza-compounds to sulfonate
derivatives17 and has for the first time been applied here to
BODIPY dyes. Cross-coupling 11 with excess 1-dimethy-
lamino-2-propyne was promoted by Pd(0) generated in situ from
(17) Kong, X.; Migneault, D.; Valade, I.; Wu, X.; Gervais, F. U.S. Patent
2007/010573, 2007.
(15) (a) Bonardi, L.; Ulrich, G.; Ziessel, R. Org. Lett. 2008, 10, 2183–
2186. (b) Cakmak, Y.; Akkaya, E. U. Org. Lett. 2009, 10, 85–88.
(16) Goze, C.; Ulrich, G.; Ziessel, R. J. Org. Chem. 2007, 72, 313–
322.
(18) Olmsted, J., III. J. Phys. Chem. 1979, 83, 2581–2584.
(19) Tleugabulova, D.; Zhang, Z.; Breqnnan, J. D. J. Phys. Chem. B
2002, 106, 13133–13138.
Org. Lett., Vol. 11, No. 10, 2009
2051

Table 1. Optical Properties of the BODIPY Derivatives
λmax,abs λmax,em Stokes shift
a
compound
solvent
(nm) (nm)
(cm-1
)
ΦF
3
PBS
PBS
523
539
307
567
0.62b
nonfl
0.15
0.10
0.40
10
10
10
10
609
nonfl
656
nonfl
372
185
393
PBS + 10% DMSO 371
664
661
666
648
PBS + 10% EtOH
368
653
374
649
DMSO
Figure 1. Absorption (light blue) and emission (light green) spectra
of 3 at 25 °C in PBS. Absorption (dark blue) and emission (dark green)
spectra of 13 at 25 °C in PBS.
12
13
15
16
CH2Cl2
PBS
CH2Cl2
PBS
543 565
521 540
521 532
522 530
717
675
396
0.54
0.78
0.52
289
396
0.61
0.71
567
-
16
EtOH
521 532
a Determined at 25 °C by using either R6G (for 3, ΦF ) 0.76 in water, ex
λ ) 488 nm) or sulfoindocyanine dye Cy 5.0 (for 10, ΦF ) 0.20 in PBS, ex
λ ) 603 nm) as standards.8a,18 All ΦF are corrected for changes in refractive
index. b QY in DMSO was found to be 0.82. c Value for a 5 × 10-6 M solution.
Note that the water-soluble BODIPY dyes 3 and 16 were
designed to have, after postsynthetic derivatization, an acid
functional group suitable for conjugation to biomolecules
through amide bond formation, and their good quantum yields
in aqueous environments are compatible with their use as
fluorescent biological labels.
In summary, the work highlights two original, tunable
synthetic methods to postsynthetically convert hydrophobic
BODIPY cores into corresponding water-soluble derivatives.
Both approaches provide hydrophilic fluorophores with a handle
(i.e., carboxylic acid or iodo functional group) for their
conjugation to various biological materials. Interestingly, the
mild conditions (i.e., NMP-water, pH 8.5, rt) of the amine/
acid coupling reaction involved in the “acylation approach” are
fully compatible with the presence of additional sophisticated
functional groups. Thus, this method in combination with
standard electrophilic sulfonation or B-F displacement reaction
has a strong potential to enhance the water solubility without
harming the optical properties of these multicomponent dyes.
Current work is directed toward introducing additional sulfonate
groups at the boron atom or on the styryl-anisole moieties of
compound 10 to disrupt its detrimental aggregation tendency
in physiological conditions.
Figure 2. Top: absorption in PBS (black line), absorption in PBS +
10% EtOH (red line), and emission (green line, exc. λ ) 522 nm, in
PBS) spectra of 16 at 25 °C. Bottom: absorption in PBS (black line),
absorption (red line), and emission (green line, exc. λ ) 603 nm)
spectra in PBS + 10% DMSO of 10 at 25 °C.
yields of 61% and 71%, respectively. Note that excitation at
567 nm does not cause any fluorescence of the dye. The
excitation spectrum of the emissive species in PBS matches
the absorption spectra of 16 in ethanol. This confirms that the
emissive species is 16.
Surprisingly, despite the presence of polysulfonated moieties,
compound 10 shows a tendency to aggregate in aqueous
solution and quench fluorescence, and the blue shift in the
absorption maximum at 609 nm is in keeping with the formation
of H-dimers19 (Figure 2). This aggregation is persistent even
at concentrations as low as 1 µM in PBS. The free fluorescent
monomer is obtained in organic solvents such as DMSO and
in PBS with 10% DMSO or ethanol. Under these conditions,
the excitation spectrum matches the absorption spectrum,
proving the absence of aggregates.
Acknowledgment. This work was supported by La Re´gion
Haute Normandie and IUF. We thank QUIDD Company for
the open access to their HPLC instruments. We also acknowl-
edge the CNRS providing research facilities. G.U. thanks ANR
JCJC05-4228 for financial support and Professor Jack Harrow-
field (ISIS in Strasbourg) for commenting on the manuscript
before publication.
Supporting Information Available: Synthetic procedures
and analytical data reported herein. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL900302N
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Org. Lett., Vol. 11, No. 10, 2009