New insights into the water-solubilisation of fluorophores by post-synthetic "click" and Sonogashira reactions

Article abstract of DOI:10.1039/c2ob25428j

New synthetic methodologies for the efficient chemical conversion of hydrophobic fluorescent dyes into bioconjugable and water-soluble derivatives are described. The combined use of an original sulfonated terminal alkyne and a metal-mediated reaction, namely the copper-catalysed Huisgen 1,3-dipolar cycloaddition ("click" reaction) or the Sonogashira cross-coupling, is the cornerstone of these novel post-synthetic sulfonation approaches.

Full text of DOI:10.1039/c2ob25428j

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New insights into the water-solubilisation of fluorophores by post-synthetic
“click” and Sonogashira reactions†
Cédrik Massif,a Sébastien Dautrey,a Alexandre Haefele,b Raymond Ziessel,b Pierre-Yves Renarda,c,d and
Anthony Romieu*a,c,d
Received 27th February 2012, Accepted 17th April 2012
DOI: 10.1039/c2ob25428j
solubilising group to synthesise hydrophilic derivatives of
various fluorescent architectures.
New synthetic methodologies for the efficient chemical con-
version of hydrophobic fluorescent dyes into bioconjugable
and water-soluble derivatives are described. The combined
use of an original sulfonated terminal alkyne and a metal-
mediated reaction, namely the copper-catalysed Huisgen 1,3-
dipolar cycloaddition (“click” reaction) or the Sonogashira
cross-coupling, is the cornerstone of these novel post-syn-
thetic sulfonation approaches.
For the majority of fluorophores belonging to the BODIPY
(and aza analogues), coumarine, cyanine, oxazine and xanthene
families, the common sulfonation reactions include: (1) electro-
philic aromatic substitution with H2SO4 (70–100%), oleum or
ClSO3H,3,4 (2) alkylation reaction with a β-haloethanesulfonic
acid or a sultone derivative,5–7 and (3) Schotten–Baumann ami-
dification reaction with an (α-sulfo-β-alanine)-based peptide
(β-Ala(SO3H)-based peptide).8 This latter one was developed by
our group and proved efficient to produce water-soluble and bio-
conjugable of a wide range of fluorescent organic dyes and
supramolecular compounds.2,7,9 Some of them, especially rhoda-
mines and red-emitting BODIPY dyes, are viable alternatives to
commercial Alexa Fluor® and CyDye™.10 In order to get more
rapidly these valuable fluorescent markers, we have recently
reported the implementation of a solid-phase version of this
Schotten–Baumann reaction using N-Fmoc-β-Ala(SO3H) as the
key building block.11 Despite these significant achievements, our
synthetic methodology is still limited to fluorophores possessing
a carboxylic acid function. To expand the scope of our “post-
synthetic” and mild sulfonation methodology to a wider range of
fluorescent architectures that do not necessarily contain func-
tional groups able to readily react with nucleophilic sulfonated
linkers, there is an overriding need for designing alternative reac-
tions allowing the grafting of sulfonated peptide-based linkers.
Since the C- and N-terminal sides of β-Ala(SO3H) polypeptides
can be easily derivatised with a terminal alkyne moiety through
peptide coupling reactions with propargylamine and propiolic
acid respectively, it seemed obvious to us that we should explore
chemical transformations involving a terminal alkyne as a versa-
tile reaction partner. Thus, we have decided to explore the poten-
tial of popular transition metal-mediated reactions, namely
copper-catalysed azide–alkyne 1,3-dipolar cycloaddition
(CuAAC, also named Huisgen–Sharpless–Meldal or “click”
reaction)12 and palladium-catalysed Sonogashira reaction,13
to achieve this ambitious goal. To the best of our knowledge,
Sonogashira cross-coupling has never been used to impart water-
solubility of fluorescent organic dyes. However, we can mention
the preliminary works from the Burgess group published in 2007
and devoted to the spectral dispersion and water solubilisation
of the tetramethyl-BODIPY system via Heck-type coupling
Introduction
It is now well established that fluorescent organic dyes are the
preferred markers for the massive number of bioanalytical and
biomedical applications whose practical implementation involves
biomolecular labelling.1 Among the numerous requirements for
an ideal fluorescent bio-label, high brightness, water-solubility,
resistance to self-aggregation and bio-orthogonal reactivity are
the most important items currently intensively explored. To
address these non-trivial issues, the most popular strategies are
based on the introduction of hydrophilic moieties (positively or
negatively charged groups, PEG-type chains and biopolymers)
and a carboxylic acid function (easily convertible in active
esters) in various positions of the polycyclic aromatic scaffolds.2
Due to its unique solubilising properties and chemical inertness
(especially in the context of bioconjugation reactions), sulfonate
(–SO3−) is probably the most popular and widely used water-
aUniversité de Rouen, Laboratory COBRA UMR 6014 and FR 3038,
IRCOF, 1 Rue Tesnière, 76821 Mont St Aignan Cedex, France.
E-mail: anthony.romieu@univ-rouen.fr; Fax: + 33 (0)2 35 52 29 71;
Tel: + 33 (0)2 35 52 24 27
bLaboratoire de Chimie Moléculaire et Spectroscopies Avancées
(LCOSA), Ecole Européenne de Chimie, Polymères et Matériaux, 25 rue
Becquerel, 67087 Strasbourg Cedex 02, France.
E-mail: ziessel@unistra.fr; Fax: (+) 33 3-68-85-27-61
cINSA de Rouen, Avenue de l’Université, 76800 St Etienne du Rouvray,
France
dCNRS Délégation Normandie, 14 Rue Alfred Kastler, 14052 Caen
Cedex, France
†Electronic supplementary information (ESI) available: Detailed syn-
thetic procedures and characterisation/spectral data for compounds 1,
5–12, 14 & 17–20. See DOI: 10.1039/c2ob25428j
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Scheme 1 Reagents and conditions: (a) Fmoc-Gly-OH (4 equiv.), DIC (2 equiv.), DMAP (0.15 equiv.), CH2Cl2–NMP (6 : 4), rt, overnight; (b)
piperidine–NMP (2 : 8), rt, 10 min; (c) Fmoc-β-Ala(SO3H)-OH (4 equiv.), BOP (4 equiv.), DIEA (12 equiv.), NMP–DMF (55 : 45), rt, overnight; (d)
propiolic acid (5 equiv.), DIC (5 equiv.), DMAP (0.3 equiv.), CH2Cl2–NMP (6 : 4), rt, overnight; (e) TFA–CH2Cl2 (1 : 1), 4 °C to rt, 1 h followed by
azeotropic evaporation, RP-HPLC purification and lyophilisation, 45% overall yield.
(C–H activation) involving an activated alkene (i.e., a Michael
acceptor bearing –CO2H or –SO3H group).14 Despite interesting
spectral properties in aqueous buffers, the corresponding deriva-
tised BODIPYs were obtained in very poor yields and no further
improvements of this synthetic methodology have been reported
to date. Conversely, “click” reaction was used intensively in the
context of fluorophores aimed at developing valuable new
approaches for challenging biological labelling and bio-sensing
applications. Thus, valuable new concepts such as “clickable”15
or “click-on”16–18 fluorophores/dyes and “click” fluorogenic
reactions19 have recently emerged. Interestingly, CuAAC has
also been implemented for the introduction of bioconjugable or
water-solubilising moieties onto hydrophobic fluorescent cores,
especially those belonging to the (aza-)BODIPY, cyanine or
squaraine rotaxane families.20 For instance, Shi et al. have
reported a colorimetric and fluorescent probe for Cu2+ and Hg2+
ions based on a distyryl BODIPY where solubility in water was
readily obtained through a classical “click” reaction between a
tetrakis-alkyne BODIPY precursor and four units of a triethylene
glycol substituted azido derivative.21
Herein, we report the transposition and practical application of
these well-known Cu(I)- and Pd(0)-catalysed reactions to the syn-
thesis of fluorophores whose bioconjugation ability and hydro-
philic character are brought by the introduction of a sulfonated
peptide-based linker. A set of structurally different fluorescent
organic dyes, including napththalene, 7-hydroxycoumarine, flu-
orescein, rhodamine 6G (R6G) and BODIPY derivatives which
cover a broad spectral range from the UV-C to the visible green
region, has been derivatised.22 The choice of these polycyclic
aromatic compounds was based on the fact that the required
azido or iodo derivatives are synthetically easily available
through literature procedures. The optical properties of the result-
ing water-soluble fluorophores were then evaluated under phys-
iological conditions to demonstrate their potential utility as bio-
labelling reagents.
we have chosen to prepare the sulfonated terminal alkyne 1,
according to a solid-phase strategy.
The experimental conditions from our previous published
study11 were used to couple the first two amino acids (glycine
and β-Ala(SO3H)) but the amidification of propiolic acid
required further optimisation (Scheme 1). Indeed, we have
observed that it is not relevant to couple this carboxylic acid
under standard conditions involving either N,N′-diisopropylcar-
bodiimide (DIC)/1-hydroxybenzotriazole (HOBt) or a phospho-
nium (or an uronium)-based peptide coupling reagent (e.g., BOP
or TSTU/HOBt),23 due to a Michael-type reaction of the released
HOBt with the activated alkyne bond, leading to the quantitative
formation of the vinyloxy-benzotriazole derivative 2. To avoid
this side-reaction, it is possible to use a non-activated alkynated
carboxylic acid analogue such as 4-pentynoic acid but this latter
will lead to a too long and flexible linker, which is sometimes
deleterious for conjugation to biomolecules/biopolymers or spec-
tral properties in aqueous environments by favoring the surfac-
tant behavior of the resulting derivatised fluorophores.
Since we assumed that this non-desired reaction would occur
with all coupling reagents bearing a masked α-nucleophile (e.g.,
N-hydroxysuccinimide (NHS), 1-hydroxy-7-azabenzotriazole
(HOAt)…), DIC was used alone but no amidification of propio-
lic acid was observed. Nevertheless, the reaction did take place
Results and discussion
The first step of the present study was devoted to the design of a
convenient synthesis of a terminal alkyne derived from β-Ala
(SO3H). Since the N-Fmoc derivative of this unusual amino acid
is fully compatible with manual or automated solid-phase
peptide synthesis (SPPS) protocols,11 we have considered the
“on-resin” derivatisation of the C-terminal side of (β-Ala
(SO3H))-containing short peptides with propiolic acid. As
we previously showed, the use of the Wang resin requires
the incorporation of a glycine spacer prior to the loading of
N-Fmoc-β-Ala(SO3H), to get good immobilisation yields. Thus,
when
a catalytic amount of N,N-dimethylaminopyridine
(DMAP) was added to the coupling mixture. The same con-
ditions have been recently reported by Cravero et al. who used
resin-bound propiolic acid and cyclohexanediones in tandem
Michael–Michael cyclisations.24 The magnetic stirring of the
heterogeneous solid–liquid reaction mixture also has a strong
influence because when the propiolic acid coupling is performed
on a large scale of resin (∼800 mg, 0.75 mmol), it is essential to
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Scheme 2 Reagents and conditions for the CuAAC reaction: “Clickable” fluorophore (1 equiv.), sulfonated terminal alkyne 1 (1.2 equiv.), CuSO4
(0.05 equiv.), sodium ascorbate (0.2 equiv.), DMSO–H2O (1 : 1), rt, overnight followed by RP-HPLC purification and lyophilisation.
Scheme 3 Reagents and conditions for the Sonogashira reaction: Iodo-fluorophore (1 equiv.), sulfonated terminal alkyne 1 (1.2 equiv.), Pd(PPh3)4
(0.1 equiv.), CuI (0.2 equiv.), DMF–H2O–Et3N (2 : 1 : 1), rt, 3 h followed by RP-HPLC purification and lyophilisation.
divide the batch of resin into three equal parts and to conduct the
reaction in three different flasks, each having a magnetic stirrer,
otherwise the reaction does not work even if a large excess of
acid is used. After cleavage from the resin using TFA–CH2Cl2
(1 : 1) and a rapid purification by reversed-phase HPLC
(RP-HPLC), compound 1 was obtained in a pure form with a
satisfactory 45% overall yield.
The availability of this unusual alkyne led us to consider its
reactivity toward various fluorescent aryl-azido derivatives 3–7
(for the synthesis of these “clickable” fluorophores, see ESI†)
under the standard conditions currently used to perform CuAAC
reaction (i.e., CuSO4 and sodium ascorbate as the catalytic
system). A mixture of DMSO–H2O (1 : 1) was used as solvent
except for BODIPY dye 5 which was found to be soluble only
in CH2Cl2–DMSO–H2O (1 : 1 : 1) (Scheme 2). This latter azido-
BODIPY was prepared from the non-isolable bromomethyl
derivative and sodium azide in DMF, itself prepared from the
methyl-BODIPY via a reaction involving N-bromosuccinimide
(NBS) in CH2Cl2 at rt (see ESI†).25 All reactions were generally
found to be complete within 12 h, and the corresponding
monosulfonated fluorophores 8–12 were recovered in a pure
form by semi-preparative RP-HPLC, with isolated yields ranging
from 35 to 55%. Their structures were confirmed by detailed
measurements, including ESI mass spectrometry and NMR ana-
lyses (see ESI†).
The practical implementation of the Sonogashira reaction with
the fluorescent aryl-iodo derivatives 13–16 (for the synthesis of
these iodo-fluorophores, see ESI†) required minor modifications
of conditions currently employed for such Pd(0)-catalysed cross-
coupling (i.e., Pd(Ph3)4 and CuI as the catalytic system). These
modifications were only focused on the reaction solvent
(Scheme 3). A mixture of DMF–H2O–Et3N (2 : 1 : 1) was the
best compromise to solubilise both the hydrophobic dye and the
hydrophilic sulfonated alkyne 1. For all aryl-iodo derivatives,
complete conversion was observed within 3 h of vigorous stir-
ring at room temperature. Conversely, it is important to note that
the same reactions performed with the aryl-bromo analogues
were always incomplete even upon further additions of alkyne 1
and catalyst. By analogy with the tetrazole-based fluorophores,
the resulting monosulfonated fluorescent derivatives 17–20 were
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Table 1 Spectral properties of water-soluble fluorophores 8–12 and 17–20 in PBS at 25 °C
a
Compound
λmax, abs/nm
ε/dm3 mol−1 cm−1
λmax, em/nm
ΦF
230
28 100
373
0.56 (0.23)b
0.02 (0.23)b
0.78 (0.76)b
0.37 (0.76)b
244
393
422
33 190
17 650
28 300
387
471
463
497
48 350
521
0.81 (0.90)b
497
66 700
526
0.60 (0.90)b
c
517
—
537
0.25 (0.78)
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Table 1 (Contd.)
a
Compound
λmax, abs/nm
ε/dm3 mol−1 cm−1
λmax, em/nm
ΦF
c
497
—
510
0.31 (0.69)
530
80 200
551
0.86 (0.88)
a See ESI† for the experimental details related to these measurements (standards, λ ex/nm…). b Quantum yield of the non-sulfonated
29
parent fluorophore: naphthalene in C6H12
,
7-hydroxycoumarine in PBS,30 fluorescein in 0.1 N NaOH,31 8-(4-iodophenyl)-BODIPYs in benzene32
and R6G in ethanol.31 c Quantity isolated was too small for a highly accurate measurement.
isolated by semi-preparative RP-HPLC (yield 40–90%)
and fully-characterised through various spectroscopic data (see
ESI†).
absence of aggregation behavior in aq. solution, the excitation
spectrum of each triazole- or alkyne-containing dye was recorded
and found to perfectly match with the absorption spectrum in all
cases (see Fig. 1 for 8–11 and ESI† for 12 and 17–20).
As expected, these novel monosulfonated derivatives were
found to be perfectly soluble in water and related aq. buffers in
the concentration range (1.0 μM to 1.0 mM) suitable for bio-lab-
elling applications, except the R6G 12 which requires the use of
DMSO as a co-solvent to prepare a homogeneous 1.0 mM stock
solution (DMSO–H2O, 2 : 8). The photophysical properties of
these novel water-soluble fluorophores 8–12 and 17–20 were
then determined under simulated physiological conditions (i.e.,
phosphate buffered saline (PBS), pH 7.5) by using UV-vis spec-
troscopy and spectrofluorimetric analysis (Table 1 and see Fig. 1
and ESI† for the corresponding absorption/excitation/emission
spectra). These results confirmed that the newly synthesised
monosulfonated derivatives behaved in the similar manner as
compared to the parent hydrophobic dyes. Indeed, molar absorp-
tion coefficients (ε) are comparable, and quantum yields (ΦF)
are good except for alkynated naphthalene 17. For this latter
compound, the dramatic reduction in fluorescence efficiency
may be attributed to the presence of an electron-deficient alkyne
at the 2-position of the naphthalene ring, as already observed for
other fluorescent dyes such as benzothiazole, coumarine and
naphthalimide derivatives.18 As a general rule and in perfect
agreement with the fluorescence behavior of “click-on” fluoro-
genic dyes recently reported in the literature,16,18 a “click” reac-
tion which adds a conjugated triazole ring onto the parent
fluorophore ring leads to a red-shift of absorption/emission
maxima and a higher quantum yield (see water-soluble naphtha-
lene and coumarine 8 and 9, entries 1 and 3 in Table 1). For the
other water-soluble fluorophores 10–12 and 19–20, the grafting
of the sulfonated alkyne 1 did not overwhelm the overall elec-
tronic structure of their fluorescent scaffolds, and molecular
motions of the resulting water-solubilising chain would cause
radiationless deactivation and partly account for the decrease of
quantum yield in aq. environment. Finally, to confirm the
Conclusions and future work
In this account, we have described the solid-phase synthesis of
an original sulfonated terminal alkyne and its potential utility in
the water-solubilisation of a variety of fluorescent dyes through
“click” and Sonogashira reactions. The versatility of this syn-
thetic approach has been demonstrated with naphthalene, cou-
marine, BODIPY, fluorescein and rhodamine dyes. Furthermore,
this is the first report focused on the implementation of popular
transition-metal catalysed reactions to graft sulfonated moieties
onto hydrophobic molecular architectures. This “post-synthetic”
sulfonation methodology might also be auspicious to enhance
the water solubility of redox active multi-component systems
such as “cascatelle” dyes26 or through-bond energy transfer cas-
settes,27 or fullerene-based nanomaterials for biomedical appli-
cations.28 For all these molecular materials carboxylic acid
functionalities are not easily available.
Acknowledgements
This work was supported by the Agence Nationale de la
Recherche (Programme Blanc 2009, ANR-09-BLAN-0081-01)
especially for a PhD grant to Cédrik Massif, (Programme
PIRIBio 2009, ANR “CLICKMASSLINK”) for a postdoctoral
fellowship to Sébastien Dautrey, and La Région Haute-
Normandie via the CRUNCh program (CPER 2007–2013). We
thank Dr. Cyrille Sabot (CNRS researcher, Bioorganic
Chemistry Team, Université de Rouen) and Nicolas Maindron
(Ph.D., Bioorganic Chemistry Team, Université de Rouen) for
achieving NMR analyses on Bruker AVANCE I 400 spectrometer
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Fig. 1 Absorption (red), excitation (green) and emission (blue) spectra of monosulfonated triazole-based fluorophores 8–11 recorded in PBS at
25 °C.
M. Leutenegger, C. Eggeling and S. W. Hell, Eur. J. Org. Chem., 2010,
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Dr Gilles Ulrich, Dr Antoinette De Nicola and Sandra Rihn from
the LCOSA for helpful discussions.
C. Massif, G. Ulrich, R. Ziessel, P.-Y. Renard and A. Romieu, Org.
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4336 | Org. Biomol. Chem., 2012, 10, 4330–4336
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