Water solubilization of xanthene dyes by post-synthetic sulfonation in organic media

Article abstract of DOI:10.1016/j.tetlet.2010.04.080

Highly water-soluble fluorescent fluorescein and rhodamine dyes were synthesized through amidification of their carboxylic acid functionality with original di- or tri-sulfonated amino linkers derived from taurine or α-sulfo-β-alanine. This post-synthetic

Full text of DOI:10.1016/j.tetlet.2010.04.080

Tetrahedron Letters 51 (2010) 3304–3308
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Water solubilization of xanthene dyes by post-synthetic sulfonation
in organic media
c
d,
c
Anthony Romieu a,b, , Delphine Tavernier-Lohr , Stéphane Pellet-Rostaing , Marc Lemaire ,
*
*
Pierre-Yves Renard a,b,e
a Equipe de Chimie Bio-Organique, COBRA-CNRS UMR 6014 & FR 3038, rue Lucien Tesnière, 76131 Mont-Saint-Aignan, France
b Université de Rouen, Place Emile Blondel, 76821 Mont-Saint-Aignan, France
c ICBMS, UMR 5246, Université Claude Bernard Lyon1, CPE, 43 Boulevard du 11 Novembre 1918, 69100 Villeurbanne, France
d ICSM, UMR 5257, Bâtiment 426, BP 17171, 30207 Bagnols Sur Ceze Cedex, France
e Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Highly water-soluble fluorescent fluorescein and rhodamine dyes were synthesized through amidifica-
tion of their carboxylic acid functionality with original di- or tri-sulfonated amino linkers derived from
Received 22 March 2010
Revised 13 April 2010
Accepted 19 April 2010
Available online 24 April 2010
taurine or
a-sulfo-b-alanine. This post-synthetic derivatization was performed in organic media both
to minimize the premature hydrolysis and to suppress the precipitation of the involved active ester of
fluorophore, frequently encountered using standard Schotten–Baumann conditions.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescein
Fluorescence
Rhodamines
Sulfonated linker
Water solubility
1. Introduction
non-fluorescent dimers and higher aggregates, especially after con-
jugation to biological material.8 Consequently, recent efforts have
Fluorescent organic dyes are widely used as non-radioactive la-
bels and as a key component of optical bio-probes for various bio-
sensing and imaging applications.1 Xanthene-based dyes such as
rhodamines2 and fluoresceins3 (and more recently rhodols4 and
rosamines5) are among the most commonly used class of fluores-
cent detection reagents. Their spectral properties, especially in
the near-infrared (NIR) range and/or in physiological conditions,
are often less optimal than those exhibited by BODIPY and cyanine
dyes. However, their use is preferred when the following valuable
properties are required: (1) high photo- and chemical stability, (2)
easy reversible formation of a colorless and non-fluorescent spiro-
cyclic structure, and (3) modulation of fluorescence properties
through reversible chemical modification (e.g., amidation or ester-
ification) of aniline (for rhodamines) or phenol (for fluoresceins)
moiety. Indeed, the latter two features are essential for the current
design of spectroscopic off-on type probes (also named fluorogenic
probes, latent or pro-fluorophores)6 used as chemo- or biosensors
for detecting various analytes (enzymes, heavy metal ions, etc.).7
For most biological applications, xanthene dyes must also exhi-
bit both good water solubility and resistance to the formation of
been devoted to the development of water-soluble analogues of
rhodamines, through the introduction of some ionizable hydro-
philic groups (carboxylic acid and/or sulfonic acid) within the xan-
thene core structure of these fluorescent dyes.9 Most of them are
commercialized by Molecular Probes (Invitrogen) and sold under
the trade name Alexa FluorÒ.10 However, such chemical functional-
ization often requires: (1) the use of aggressive reagents such as
oleum or concentrated sulfuric acid often not compatible with
the stability of the fluorophore, or (2) restarting the synthesis of
the fluorescent core from the beginning with building blocks bear-
ing the water-solubilizing groups (de novo approach). Thus, these
synthetic processes often lead to the desired fluorophore in a mod-
est overall yield and are not amenable to large-scale preparation
(i.e., gram scale). Recently, we have reported a straightforward
method to enhance the water solubility of organic dyes (BODIPYs,
cyanines and rhodamines) by a post-synthetic chemical derivatiza-
tion of their carboxylic acid functionality with a poly-sulfonated
peptide-based linker (i.e.,
a-sulfo-b-alanine di- or tripeptide) un-
der standard Schotten–Baumann conditions.11,12 This methodology
has not yet been applied to the fluorescein derivatives and high
hydrophobic extended conjugated rhodamines. However, the cor-
responding water-soluble analogues of these latter long-wave-
length fluorophores could be useful as laser dyes and fluorescent
* Corresponding authors. Tel.: +33 2 35 52 24 15; fax: +33 2 35 52 29 71 (A.R.).
E-mail address: anthony.romieu@univ-rouen.fr (A. Romieu).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.080

A. Romieu et al. / Tetrahedron Letters 51 (2010) 3304–3308
3305
1. CH3CN, oleum, 4ºC then reflux
markers in numerous bio-labeling, bio-imaging, and single-mole-
cule-based spectroscopy applications.
In this Letter, we report the water-solubilization of fluorescein,
benzo[b]thiophene-, and indole-containing rhodamine dyes,
through amidification of their carboxylic acid function with 2-ami-
2. H2O, 4ºC then reflux
O
SO3H
SO3H
2 (Bu3N salt), 55%
3. Dowex H+ 50WX-8
4. Bu3N, lyophilization
H2N
OH
acrylic acid
noethane-1,1-disulfonic acid and tripeptide (
a
-sulfo-b-alanine)3,
modified Ritter
reaction
sulfonation followed
by acetamide hydrolysis
respectively. The use of tributylammonium salts of these two
poly-sulfonated linkers was considered in order to perform the
derivatization reaction in organic media (instead of a mixture of
DMF and aq buffer), especially to prevent both the premature
hydrolysis and precipitation of the involved N-hydroxysuccinimide
(NHS) active ester of hydrophobic rhodamine dyes, previously ob-
served by us when the reaction was conducted under standard
Shcotten–Baumann conditions. The spectral properties of the
resulting water-soluble xanthene dyes were then evaluated under
physiological conditions.
O
O
O
S
O
decarboxylation
O
O
N
OH
H
H
- CO2
N
H
SO3H
SO3H
1
Scheme 1. Synthesis of 2-aminoethane-1,1-disulfonic acid and proposed mecha-
nism for its formation.
O
O
HO
2. Results and discussion
O
2.1. Synthesis of water-soluble fluorescein derivative 4
N
OH
First, we focused on the water-solubilization of fluorescein. To
our knowledge, only the di-sulfonated rhodamine derivative devel-
oped by Molecular Probes and named Alexa FluorÒ 488 is currently
used as an hydrophilic substitute of this xanthene dye. In order to
offer an alternative to this expensive commercial fluorescent mar-
ker, we have explored the post-synthetic sulfonation of xantha-
mide derivative 3 with the original di-sulfonated amino linker 2
(Scheme 2). In the present case, it is not essential to use a water-
solubilizing linker bearing a terminal reactive group for bioconju-
gation because the ultimate goal of this synthesis was to provide
a new phenol-based fluorophore useful for the preparation of fluo-
rogenic probes based on the pro-fluorescent concept (involving the
reversible modification of its phenol moiety). The use of a fluores-
cein derivative bearing a tertiary amide of isonipecotic acid at the
20-position instead of the current carboxyl group was preferred to
provide steric protection and so to improve the photostability of
fluorescein.13 Furthermore, this is the most common way to avoid
the undesired cyclization equilibrium leading to the formation of
the colorless and non-fluorescent spirolactone (especially at pH
<6). Synthesis of 2-aminoethane-1,1-disulfonic acid 2 was accom-
plished in one step from acrylic acid, oleum, and acetonitrile using
3
O
1. 2 (TBA salt), BOP, DIEA, DMF
2. RP-HPLC
3. Dowex H+
O
O
HO
O
N
SO3H
H
N
SO3H
O
4 (acid form), 40%
Scheme 2. Synthesis of water-soluble fluorescein 4.
tively. The introduction of the di-sulfonated linker onto the isonip-
ecotic acid arm of fluorescein 4 was confirmed by 1H NMR and ESI
mass analyses.
the conditions reported by Wagner et al. for the preparation of a-
sulfo-b-alanine through a modified Ritter reaction (Scheme 1).14
Yet, in our hands, such experimental conditions did not provide
this amino acid but the taurine-like di-sulfonated linker 2. Since
the heating temperature of the sulfonation reaction mixture was
not indicated by Wagner et al. in their publication (dating from
1968), we chose to heat the reaction mixture under reflux and
we suspect that under those harsh experimental conditions, once
2.2. Synthesis of water-soluble far-red emitting rhodamine
derivatives 12 and 13
To demonstrate the full potential of this water-solubilizing pro-
cedure performed in organic media, the post-synthetic sulfonation
of benzo[b]thiophene- and indole-containing rhodamine dyes 5
and 6 was also investigated. These two compounds belong to a no-
vel class of highly fluorescent rhodamine derivatives with long-
wavelength absorption and emission, reported in 2003 by Liu
et al.16 We have recently developed an alternative pathway to
these valuable far-red emitting fluorophores, requiring an aryl–
aryl coupling through C–H activation followed by a Pictet–Spingler
reaction, to facilitate the synthetic access to these new rigid rhoda-
mine derivatives in fewer steps.17 Our preliminary experiments
aiming at the post-synthetic derivatization of these fluorophores
with poly-sulfonated amino linkers under previously reported
standard Schotten–Baumann conditions failed. This was due to
the complete precipitation of the corresponding NHS esters in
the mixture of DMF and aq buffer. Furthermore, the small amounts
of derivatized fluorophores that we obtained showed that their
great hydrophobic character could not be countered by the intro-
duction of only two sulfonate groups.
N-acetyl derivative of a-sulfo-b-alanine 1 was formed by the mod-
ified Ritter reaction, a spontaneous decarboxylation followed by
the sulfonation reaction occurred, leading to 2 (Scheme 1). The
structure of 2 was confirmed by detailed measurements, including
ESI mass spectrometry and NMR analyses. This compound was
converted into the corresponding tributylammonium salt (TBA
salt) and dissolved in dry N-methylpyrrolidone (NMP) to yield a
stock solution (0.5 M) suitable for post-synthetic derivatizations
in organic media.
Thereafter, di-sulfonated amino linker 2 could be readily cou-
pled to fluorescein derivative 3 by using BOP phosphonium salt15
and DIEA in dry DMF to give the desired water-soluble analogue
4 in a satisfying yield (40%). Purification and desalting of 4 were
sequentially achieved by semi-preparative reversed-phase HPLC
(RP-HPLC) and ion-exchange chromatography (Dowex H+), respec-

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A. Romieu et al. / Tetrahedron Letters 51 (2010) 3304–3308
Consequently, conjugation of these rhodamines to tri-sulfo-
nated linker 7 in organic media was considered. This -sulfo-b-ala-
main unavoidable drawback of reactions performed at the micro-
a
mole scale. The structures of 12 and 13 were unambiguously con-
firmed by ESI mass analyses and their purity (>95%) was checked
by RP-HPLC.
nine tripeptide was easily prepared on a gram scale according to
standard peptide synthesis protocols (Fmoc strategy).11 Its conver-
sion into TBA salt was achieved using the protocol applied to 2-
aminoethane-1,1-disulfonic acid 2, and a 0.35 M solution of the
organic solubilized tri-sulfonated linker in dry NMP was prepared.
Since the sulforhodamine sulfonyl chlorides are of delicate use for
direct conjugation to amine-containing compounds, we first con-
sidered the conversion of 5 and 6 into their carboxylic acid deriv-
atives 8 and 9, respectively, through the introduction of a glycine
spacer. Indeed, the activation of these latter reactive dyes is more
convenient and the small-scale implementation of the subsequent
coupling reaction (less than 10 mg of starting reactive fluorophore)
with the valuable tri-sulfonated linker 7 will be easier because the
corresponding NHS ester is less sensitive to moisture than the sul-
fonyl chloride derivative. Thus, rhodamine carboxylic acids 8 and 9
were obtained using the three-step procedure reported by us for
sulforhodamine 10112 (Scheme 3). Thereafter, 8 and 9 were quan-
titatively converted into the corresponding NHS esters 10 and 11,
respectively, by treatment with the peptide coupling uronium re-
agent TSTU and DIEA in dry NMP. Finally, the crude mixture of
NHS ester 10 (or 11) was subjected to aminolysis with tripeptide
2.3. Photophysical properties of the synthesized water-soluble
fluorescent dyes
As expected, fluorescein and rhodamine derivatives 4, 12, and
13 were found to be perfectly soluble in water and related aq buf-
fers in the concentration range (1 lM to 10 mM) suitable for bio-
labeling applications. The photophysical properties of these three
novel water-soluble fluorophores were evaluated under simulated
physiological conditions (i.e., phosphate buffered saline (PBS), pH
7.5) and collected in Table 1. As would be expected from previous
studies, the presence of a hydrophilic linker on the peripheral ben-
zene ring does not affect the spectral properties for the dyes except
for the indole-based rhodamine 13 (Fig. 1). Indeed, molar absorp-
tion coefficients (e) are comparable to those of the parent fluoro-
phores, and quantum yields (UF) are good especially for the
water-soluble rhodamine emitting beyond 600 nm. Interestingly,
the value of brightness (
e
ꢀ
UF) for 12 (39,600 Mꢁ1 cmꢁ1) is of
the same order as those reported for sulfoindocyanine dyes Cy
5.018 (50,000 Mꢁ1 cmꢁ1) and Cy 5.519 (43,700 Mꢁ1 cmꢁ1), the most
popular water-soluble NIR fluorophores for bio-imaging applica-
tions. The excitation spectrum of 12 matches its absorption spec-
trum (see supplementary material).This confirms the absence of
H-aggregates in this case. Surprisingly, despite the presence of
the same tri-sulfonated tail, 13 shows a tendency to aggregate in
aqueous solution, which issued in a quenching of its fluorescence
(Fig. 1b). The observed blue shift in the absorption maximum at
602 nm is in keeping with the formation of H-dimers.19 This aggre-
(a-sulfo-b-alanine)3 in the presence of DIEA to give the water-sol-
uble rhodamine 12 (or 13). RP-HPLC purification using triethylam-
monim bicarbonate (TEAB) buffer and acetonitrile as eluents,
followed by desalting on ion-exchange resin Dowex H+ provided
12 and 13 in a pure form (mixture of two regioisomers, each con-
sisting of four racemic diastereomers, isolated yield: 45% for 12
and 35% for 13). This non-quantitative yield for the present post-
synthetic sulfonation although it was carried out in an organic
medium was explained both by the moderate reactivity of NHS es-
ter toward tri-sulfonated amino linker (steric hindrance?) and by
significant losses of derivatized fluorophore during the chromato-
graphic purification and lyophilization steps. Indeed, this is the
gation is persistent even at concentrations as low as 1 lM in PBS.
The differences in aggregation ability that we observed between
12 and 13 are probably due to the nature and orientation of
N+
N
O
N+
N
O
Ar
Ar
2
Ar
Ar
3 steps (Romieu et al. 2008)
SO3-
SO3-
SO2
OH
N
H
O
SO3H
S
4
N
8, Ar = benzothiophene
9, Ar = indole
=
or
Ar
Ar = benzothiophene
Ar = indole
5, Ar = benzothiophene
6, Ar = indole
TSTU, DIEA, NMP
N+
N
O
N+
N
O
SO3H
SO3H
SO3H
H
N
H
N
OH
H2N
Ar
Ar
Ar
2
Ar
2
O
O
O
O
7 (TBA salt)
SO3-
SO2
SO3-
SO2
O
O
SO3H
O
N
SO3H
O
SO3H
O
1. 7, DIEA, NMP
2. RP-HPLC
N
H
N
H
H
N
H
N
OH
HN
O
3. Dowex H+
O
4
4
12 (acid form), Ar = benzothiophene, 45%
13 (acid form), Ar = indole, 35%
10, Ar = benzothiophene, quant. yield
11, Ar = indole, quant. yield
Scheme 3. Synthesis of water-soluble rhodamines 12 and 13.

A. Romieu et al. / Tetrahedron Letters 51 (2010) 3304–3308
3307
Table 1
Spectral properties of water-soluble xanthene dyes in physiological conditions
(Mꢁ1 cmꢁ1
)
UF
a
Dye
Solvent
kmax,
(nm)
kmax,
(nm)
em
Stokes shift (nm)
e
abs
4
12
13
13
PBS
PBS
PBS
499
628
602, 643
650
522
645
Nonfl
672
23
17
Nonfl
22
59,000
90,000
0.69
0.44
Nonfl
0.19
b
—
b
PBS + 5% (w/v) BSA
—
a
Determined at 25 °C by using either fluorescein (for 4, UF = 0.90 in 0.1 M NaOH, Ex. k = 488 nm) or sulfoindocyanine Cy 5.0 (for 12 and 13, UF = 0.20 in PBS, Ex. k = 600 nm)
as standards.18,24 All UF are corrected for changes in refractive index.
b
Quantity isolated was too small for a highly accurate measurement.
heterocycle (benzo[b]thiophene or N-methylindole) fused to the
xanthene ring. The free fluorescent monomer of 13 is obtained in
PBS using 5% (w/v) bovine serum albumin (BSA), an additive often
used in buffers for mimicking body fluids (Fig. 1c). The strong
enhancement in emission in conjunction with the red shift in the
absorption maximum in the presence of BSA observed for 13 points
to dye-BSA interactions. The beneficial effect of such dye-protein
interactions on quantum yield has been already reported for series
of cyanine labels commercialized by Dyomics.20
2.4. Alternative synthetic route to water-soluble rhodamine 6G
(R6G-WS)
In addition to these promising results, we have also evaluated
the positive effect (or not) on the reaction yield for the post-syn-
thetic sulfonation performed in an aprotic polar solvent instead
of a mixture of NMP and aq buffer as previously described. To this
end, rhodamine 6G (R6G) carboxylic acid 14 was chosen as a model
xanthene dye. Since, this compound is significantly less hydropho-
bic than benzo[b]thiophene- and indole-containing rhodamine
dyes, its derivatization with dipeptide (a-sulfo-b-alanine)2 15
was considered (Scheme 4).12 Thus, after the conversion of 14 into
NHS ester, the crude mixture was divided into two equal parts and
aminolysis with di-sulfonated amino linker was carried either in
NMP (with the corresponding TBA salt of 15) or in a mixture of
NMP and borate buffer (0.1 M, pH 8.2). After RP-HPLC purification,
desalting, and lyophilization, the recovered amount of R6G-WS is
2.7-fold higher for the reaction performed under non-aqueous con-
ditions. This result clearly shows that the modifications brought to
the original post-synthetic sulfonation procedure published in
200812 are effective to prevent competitive hydrolysis of fluoro-
phore active ester and so to improve the yield of this water-solubi-
lization process.
Br
O
N+
N
H
O
H
O
OH
O
14
SO3H
O
SO3H
O
H
N
1. TSTU, DIEA, NMP, quant. yield
2a. 15 (Et2NH salt), aq. borate buffer (pH 8.2), 4°C to rt
or 2b. 15 (TBA salt), NMP, 4ºC to rt
H2N
OH
15
N
O
N+
H
H
SO3H
O
SO3-
O
O
H
N
H
N
OH
O
Figure 1. (a) Normalized UV–vis absorption (—) and emission (- - -) spectra of (Ex.
k = 605 nm) 12 in PBS at 25 °C. (b) Normalized UV–vis absorption (—) and emission
(- - -) spectra of (Ex. k = 605 nm) 13 in PBS at 25 °C. (c) Normalized UV–vis
absorption (—) and emission (- - -) spectra of (Ex. k = 600 nm) 13 in PBS + 5% BSA
(w/v) at 25 °C.
O
R6G-WS (acid form), 38% (2a) and quant. yield (2b)
Scheme 4. Synthesis of R6G-WS.

3308
A. Romieu et al. / Tetrahedron Letters 51 (2010) 3304–3308
5. For recent examples, see: (a) Wu, L.; Burgess, K. Org. Lett. 2008, 10, 1779–
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Acknowledgments
This work was supported by La Région Haute Normandie via the
CRUNCh program (CPER 2007–2013) and IUF. We thank Dr. G.
Clavé for the synthesis of sulfoindocyanine dye Cy 5.0 (used as
standard for QY measurements) and purification of R6G-WS, and
QUIDD company for the open access to their HPLC instruments.
18. Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S.
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Supplementary data
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Supplementary data (detailed synthetic procedures, spectro-
scopic and photophysical characterizations of water-soluble xan-
thene dyes 4, 12 and 13) associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2010.04.080.
References and notes
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