7-Hydroxycoumarin-hemicyanine hybrids: A new class of far-red emitting fluorogenic dyes

Article abstract of DOI:10.1021/ol801582w

(Chemical Equation Presented) The design and synthesis of novel water-soluble far-red emitting phenol-based fluorophores derived from 7-hydroxycoumarin are described. These hemicyanine-coumarin hybrids display promising spectroscopic features such as large apparent Stokes shift (ranging from 60 to 140 nm) and fluorescence emission maxima between 620 and 720 nm in physiological conditions. Their utility was then illustrated by the preparation of an original fluorogenic probe of penicillin G acylase (PGA) whose fluorescence is unveiled through an enzyme-initiated domino reaction.

Full text of DOI:10.1021/ol801582w

ORGANIC
LETTERS
2008
Vol. 10, No. 19
4175-4178
7-Hydroxycoumarin-Hemicyanine
Hybrids: A New Class of Far-Red
Emitting Fluorogenic Dyes
Jean-Alexandre Richard,†,‡ Marc Massonneau,‡ Pierre-Yves Renard,*,†,§ and
Anthony Romieu*,†,§
Equipe de Chimie Bio-Organique, UniVersite´ de Rouen, Place Emile Blondel,
76821 Mont-Saint-Aignan, France, UMR CNRS 6014, COBRA, IRCOF, rue Lucien
Tesnie`re, 76130 Mont-Saint-Aignan, France, and QUIDD, Technopoˆle du Madrillet,
50, rue Ettore Bugatti, 76800 Saint-Etienne-du-RouVray, France
pierre-yVes.renard@uniV-rouen.fr; anthony.romieu@uniV-rouen.fr
Received July 11, 2008
ABSTRACT
The design and synthesis of novel water-soluble far-red emitting phenol-based fluorophores derived from 7-hydroxycoumarin are described.
These hemicyanine-coumarin hybrids display promising spectroscopic features such as large apparent Stokes shift (ranging from 60 to 140
nm) and fluorescence emission maxima between 620 and 720 nm in physiological conditions. Their utility was then illustrated by the preparation
of an original fluorogenic probe of penicillin G acylase (PGA) whose fluorescence is unveiled through an enzyme-initiated domino reaction.
During the past two decades, intensive research efforts
have been devoted to the development of numerous enzyme
assays for various bioanalytical and biological applications
including high-throughput screening or biomolecular imag-
ing.1 The use of spectroscopic probes whose fluorescence
properties dramatically change upon reaction, covalent
interaction, or reversible binding with target analytes is often
required. Indeed, molecular fluorescence spectroscopy is one
of the most powerful and simple methods to visualize in Vitro
and in ViVo biochemical and cell biological processes in a
real-time manner, with a high degree of accuracy, and by
using simple instruments and facilities.2 Thus, numerous
profluorescent probes that unmask their intense fluorescence
only by a user-designated chemical reaction have been
elaborated for the visualization of biologically relevant
molecules (metal cations, reactive oxygen species, thiols)3
and the detection of various enzymes like esterases, phos-
phatases, proteases, or ꢀ-galactosidase.4
Recently, the research of optical bioprobes absorbing and
emitting in the near-infrared (NIR) region has gained a huge
interest.5 Indeed, light in the region 650-900 nm is poorly
(2) Haugland, R. P.; Spence, M. T. Z.; Johnson, I. D.; Basey, A. The
Handbook: A Guide to Fluorescent Probes and Labelling Technologies,
10th ed.; Molecular Probes; Eugene, OR, 2005. de Silva, A. P.; Gunaratne,
H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher,
J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515–1566.
(3) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem.
Soc. ReV. 2008, 37, 1465–1472. Chen, X.; Wang, X.; Wang, S.; Shi, W.;
Wang, K.; Ma, H. Chem.sEur. J. 2008, 14, 4719–4724. Miller, E. W.;
Chang, C. J. Curr. Opin. Chem. Biol. 2007, 11, 620–625. Li, X.; Zhang,
G.; Ma, H.; Zhang, D.; Li, J.; Zhu, D. J. Am. Chem. Soc. 2004, 126, 11543–
11548.
† COBRA-CNRS UMR 6014.
‡ QUIDD (QUantitative Imaging in Drug Development).
§ Universite´ de Rouen.
(1) Johnsson, N.; Johnsson, K. ACS Chem. Biol. 2007, 2, 31–38.
Goddard, J. P.; Reymond, J. L. Curr. Opin. Biotechnol. 2004, 15, 314–
322.
(4) Chen, X.; Sun, M.; Ma, H. Curr. Org. Chem. 2006, 10, 477–489.
Takakusa, H.; Kikuchi, K.; Urano, Y.; Kojima, H.; Nagano, T. Chem.sEur.
J. 2003, 9, 1479–1485.
10.1021/ol801582w CCC: $40.75
Published on Web 09/03/2008
2008 American Chemical Society

absorbed by biomolecules and thus can penetrate more deeply
into tissues (up to several centimeters).6 Autofluorescence
is also minimized in this region as well as Rayleigh-Tyndall
light scatttering artifacts.7 Thus, much higher signal-to-noise
ratios can be obtained, and the corresponding NIR fluorescent
probes are expected to be suitable for emerging and chal-
lenging biomedical applications such as in ViVo diagnostic
imaging.
However, we found that thiols have a deleterious quenching
effect on the fluorescence of these fluorophores thus prevent-
ing their use in imaging applications of living systems.11 To
circumvent this issue, we decided to explore a novel class
of NIR dyes derived from 7-hydroxycoumarin that do not
contain a thiol-sensitive quinoimine ring. Herein, we report
the synthesis of these water-soluble far-red emitting phenol-
based fluorophores, their promising spectral properties in
physiological conditions (large Stokes shift and fluorescence
emission maxima between 620 and 720 nm) and their
application for the preparation of latent fluorescent probes
aimed at the detection of the model protease, PGA.
The most popular synthetic strategy to obtain NIR fluo-
rophores consists of the extension of the π-π conjugation
system of conventional dyes (i.e., cyanine dyes, fluorescein,
rhodamines, and BODIPYs) but such chemical modification
often leads to molecules poorly or not soluble in aqueous
media, sometimes chemically unstable (especially for the
long polymethine chain cyanine dyes such as Cy 7.0 and its
analogues) and/or highly sensitive to photobleaching.8 Fur-
thermore, few water-soluble NIR dyes having a reactive
group (i.e., an aniline, a phenol, or a thiophenol moiety)
whose reversible chemical modification (e.g., acylation,
alkylation) significantly affects their fluorescence properties
have been reported to date. The chemistry of such fluorogenic
dyes was recently investigated by the group of Weissleder
through the development of original Nile Blue analogues (i.e.,
benzo[a]phenoxazine dyes) and new symmetric and asym-
metric xanthene dyes (i.e., naphthofluorescein and rhodan-
aphthofluor derivatives).9 The potential utility of their
disulfonated benzo[a]phenoxazine scaffold (2SBPO) was
illustrated by the preparation of fluorogenic probes suitable
for the in ViVo imaging of two different hydrolytic enzymes:
ꢀ-galactosidase and dipeptidyl peptidase IV.10 Given their
critical role in numerous diseases, the detection of specific
protease activities in ViVo is of great importance, especially
for diagnostic and therapeutic purposes. In that context, we
recently reported original profluorescent substrates of penicil-
lin G acylase (PGA) and caspase-3, whose strong fluorescent
7-hydroxycoumarin dye is released through enzyme-initiated
domino reactions.11 Such a strategy requires the introduction
of a self-immolative spacer (e.g, p-aminobenzyl alcohol,
PABA) between the peptidyl substrate and the fluorophore
that results in many beneficial effects such as higher stability
in physiological media as well as better enzymatic recogni-
tion and cleavage kinetics. In order to extend this approach
to the NIR range, we have applied the same synthetic strategy
to the red-emitting acridinone and oxazinone phenol dyes
already reported in the literature (i.e., DAO and resorufin).
The targeted fluorogenic phenol dyes were designed as
follows: (1) the pro-fluorescence properties should be
obtained taking into account that 7-alkoxycoumarins emit
weakly compared to the phenol free 7-hydroxycoumarins;12
(2) a push-pull device was designed between the phenol
functionality of the 7-hydroxycoumarin and an indolium
moiety; (3) the red-shift of the wavelength emission should
be obtained by the introduction of one or two double bonds
in the 3-position of the coumarin, thus extending the
π-conjugation of the resulting dye; (4) water solubility of
the fluorophore should be obtained through to the introduc-
tion of up to three sulfonate groups onto the indolium moiety
(Figure 1).
Figure 1. General structure of the targeted water-soluble far-red
(5) Kiyose, K.; Kojima, H.; Nagano, T. Chem.-Asian J. 2008, 3, 506–
515.
emitting phenol-based fluorophore.
(6) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123–128.
(7) Licha, K. Top. Curr. Chem. 2002, 222, 1–29.
(8) Toutchkine, A.; Nguyen, D.-V.; Hahn, K. M. Org. Lett. 2007, 9,
2775–2777.
Fluorophores 4a-e were synthesized Via a base-mediated
electrophilic substitution of indolium derivatives 3a-c13 as
depicted in Scheme 1. First, the reaction with 3-formylcou-
marin 1 in ethanol in the presence of pyrrolidine gave the
water-soluble hemicyanine-coumarin hybrids 4a, 4c, and
4e in good yields. Interestingly, the removal of the acetyl
(9) Hilderbrand, S. A.; Weissleder, R. Tetrahedron Lett. 2007, 48, 4383–
4385. Ho, N.-H.; Weissleder, R.; Tung, C.-H. Tetrahedron 2006, 62, 578–
585.
(10) Lai, K. S.; Ho, N.-H.; Cheng, J. D.; Tung, C.-H. Bioconjugate
Chem. 2007, 18, 1246–1250. Ho, N.-H.; Weissleder, R.; Tung, C.-H.
ChemBioChem 2007, 8, 560–566. Ho, N.-H.; Weissleder, R.; Tung, C.-H.
Bioorg. Med. Chem. Lett. 2006, 16, 2599–2602.
(11) Richard, J.-A.; Meyer, Y.; Jolivel, V.; Massonneau, M.; Dumeunier,
R.; Vaudry, D.; Vaudry, H.; Renard, P.-Y.; Romieu, A. Bioconjugate Chem
2008, 19, 1707–1718. Meyer, Y.; Richard, J.-A.; Massonneau, M.; Renard,
P.-Y.; Romieu, A. Org. Lett. 2008, 10, 1517–1520.
(12) Setsukinai, K.-I.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Nagano, T.
J. Chem. Soc., Perkin Trans. 2 2000, 2453–2457.
(13) For the synthesis of 3a–c, see the Supporting Information.
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Org. Lett., Vol. 10, No. 19, 2008

Scheme 1
.
Synthesis of Water-Soluble Far-Red Emitting
Phenol-Based Fluorophores 4a-e
Table 1. Absorption and Fluorescence Data for
Hemicyanine-Coumarin Hybrids
b
λmax(abs) λmax(em) Stokes shift
εa
ΦF
dye
(nm)
(nm)
(nm)
(×104) (%) solvent
4a
555
630
623
564
491
573
485
630
592
494
508
578
495
643
620
649
642
720
653
636
604
648
722
655
654
643
625
659
65
19
19
139
162
63
119
18
130
161
146
65
130
16
2.28
2.22
2.20
2.70
2.45
7.04
3.55
7.80
2.45
2.35
2.50
6.55
3.37
4.18
0.7 PBSc
2.3 DMSO
4.9 ethanol
1.3 PBS
1.3 DMSO
1.3 PBS
1.2 DMSO
3.1 ethanol
1.1 PBS
1.7 DMSO
1.0 ethanol
2.7 PBS
4b
4c
4d
4e
2.5 DMSO
1.4 ethanol
a Molar extinction coefficient are in M-1 cm-1
quantum yields were determined at 25 °C by using rhodamine 6G (ΦF
90% in water at 25 °C) or sulfocyanine dye Cy 5.0 (ΦF ) 20% in PBS at
25 °C) as standards.15 c PBS ) 100 mM phosphate buffer + 150 mM NaCl,
pH 7.4.
.
b The fluorescence
)
of photons and a diminution of the fluorophore self-quenching.
This might be attributed to an excited-state intramolecular
charge transfer (ICT) between the phenolate group (donor) and
the indolium moiety (acceptor) within the same dye molecule.
Furthermore, a marked negative solvatochromism in the absorp-
tion spectrum of dimethine hemicyanine-coumarin hybrids 4a,
4c, and 4e (∼60 nm shift from PBS to ethanol) was observed
with increasing polarity of the solvents, but no apparent
solvatochromism in the emission spectra was detected. These
properties are due to hydrogen-bonding interaction between the
solvents and the dye molecule and proved that an excited-state
ICT should take place in the present case.14 A slight improve-
ment of the quantum yields was observed along with the
increase in the number of negatively charged sulfonate groups
directly attached to the (benzo)indole ring. Indeed, this hydro-
solubilization strategy enabled reducing dye-dye interactions
in water because of Coulombic repulsion and partially prevents
static quenching through association. Brightness (ΦFε) values
in physiological conditions (e.g., 1770 for 4e in PBS) are
comparable to those determined for the commercially available
red-emitting acridinone or oxazinone phenol dyes (e.g., 1930
for DAO in PBS).11
group of phenol moiety occurred simultaneously during this
base-mediated condensation reaction. In order to shift the
fluorescence emission to longer red wavelengths, an ad-
ditional double bond was added to the coumarin core of 1
by means of a Wittig reaction and afforded 3-cinnamyl
coumarin 2 in 60% yield (see the Supporting Information).
This latter aldehyde was then condensed with indolium
derivatives 3a,b under the same conditions as used for 1.
However, the corresponding fluorophores 4b and 4d were
obtained in lower yields (30-35%) than the dimethine dyes.
These modest yields were explained by a competitive
pyrrolidine-catalyzed retroaldol reaction of 2 transitorily
leading to the 3-formylcoumarin 1 which subsequently reacts
with indolium to give 4a or 4c. All spectroscopic data (see
the Supporting Information), especially NMR and mass
spectrometry, were in agreement with the structures assigned.
As expected, fluorophores 4a-e were found to be perfectly
soluble in water and related aqueous buffers in the range of
concentrations suitable for biomolecular labeling applications
(1.0 µM to 5 mM). The inertness of these fluorescent phenols
toward thiols was also demonstrated through incubation with
1,4-dithioerythritol (DTE) and subsequent LC-MS analyses
(see the Supporting Information).
The photophysical properties of the five novel water-soluble
phenol-based fluorophores available were evaluated in various
solvents (i.e., PBS and polar organic solvents) and collected in
Table 1 (for their normalized absorption and emission spectra,
see the Supporting Information). As expected, the extension of
the aromatic system conjugation of parent 7-hydroxycoumarin
resulted in a dramatic red-shift of the emission maxima beyond
600 nm. Interestingly, compounds 4a-e exhibited a large Stokes
shift (ranging from 60 to 140 nm according to the solvent and
the length of polymethine chain) which is beneficial for a better
detection sensitivity thanks to the decrease of the reabsorption
Several possible hypotheses to explain the relatively modest
fluorescence efficiency of phenol dyes 4a-e can be found:
dynamic quenching of excited dye molecules through collision
with dye in the ground-state and excited-state twisting (rotation/
vibration) of the central tetramethine bridge separating the two
(hetero)cyclic heads (“loose belt effect”)16 or static quenching
through association (other than H-aggregation) of ground-state
(14) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y.
J. Am. Chem. Soc. 2005, 127, 4170–4171.
(15) Magde, D.; Wong, R.; Seybold, P. G. Photochem. Photobiol. 2002,
75, 327–334. Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.;
Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105–111.
Org. Lett., Vol. 10, No. 19, 2008
4177

dye molecules. Thus, current work is ongoing in our laboratory
to rigidify these pseudocyanines in order to improve the
quantum yields in physiological conditions.
mediated condensation reaction with aldehyde 6. Spectroscopic
measurements performed in PBS at pH 7.5 revealed that the
etherification of the 7-OH group of 4c effectively quenched its
fluorescence at 636 nm (Figure 2B).17 This particular trend
confirmed that hemicyanine-coumarin hybrids 4a-e can be
used in the design and synthesis of fluorogenic probes sensitive
to various analytes. Finally, profluorophore 7 was tested against
PGA. Figure 2C showed the time-course for the amidase-
catalyzed hydrolysis of 7. After PGA was added to the substrate
solution, a strong fluorescence signal generated at 636 nm
indicated the catalytic cleavage of the carboxamide bond and
the release of the free fluorescent phenol 4c. Furthermore, no
nonspecific cleavage of the probe was detected in a control
reaction where 7 was incubated only in phosphate buffer.
Overall, these results confirm that the latent red fluorophore 7
is a suitable substrate for penicillin amidase. Thus, in addition
to the profluorescent character of our phenol-based fluorophores,
the full stability of fluorogenic probe 7 as well as its suitability
for the detection of a model protease have been shown.
In summary, we have designed a novel class of water-soluble
far-red emitting phenol-based fluorophores by coupling a
polysulfonated (benzo)indolium unit to the 3-formyl (or the
3-cinnamyl) derivative of umbelliferone. Contrary to the com-
mercially available red-emitting acridinone and oxazinone
phenol dyes, these fluorescent labels are not reactive toward
biological thiols. They exhibit a large Stokes shift, and the
emission maximum wavelength can be easily tuned between
620 and 720 nm thanks to the extension of the electron density
to the indole moiety Via a push-pull device. Furthermore, we
confirmed the profluorescent features of our pseudocyanine dyes
through the synthesis and in Vitro validation of the PGA
sensitive fluorogenic probe 7. In order to expand the scope of
these promising latent far-red fluorophores to the visualization
of various biologically relevant molecules and activities inside
living cells, further works are in progress to improve both
the brightness of these fluorescent phenols and their cell
permeability by using bioactivatable masking groups for the
negative charges of sulfonate moieties.18
Prior to the use of these novel phenol-based fluorophores in
the context of fluorogenic probes based on the pro-fluorescent
concept, it was essential to check that the chemical modification
of their hydroxyl group (i.e., modification of the phenol either
to an ether or an ester) effectively quenched their red fluores-
cence emission. For this purpose, we synthesized a fluorogenic
probe suitable for the detection of model amidase PGA. We
chose to use hemicyanine-coumarin hybrid 4c as the fluores-
cent phenol because it represented a good compromise between
high molar extinction coefficient, acceptable quantum yield and
ease of synthesis. Thus, our synthetic strategy to the PGA
sensitive profluorescent probe 7 involved the introduction of
Acknowledgment. This work was supported by La Re´gion
Haute-Normandie, QUIDD, and Institut Universitaire de
France (IUF). We thank Elisabeth Roger (INSA de Rouen)
for IR measurements and Dr. Christophe Portal (QUIDD)
for English correction of the manuscript.
Figure 2. (A) Synthesis of the PGA sensitive fluorogenic probe 7.
(B) Emission spectra (ex λ ) 560 nm) of pseudocyanine dye 4c
(red line) and fluorogenic probe 7 (orange line) at 37 °C in
phosphate buffer (concentration 3.9 µM). (C) Fluorescence emission
time-course of probe 7 (concentration 3.9 µM) with PGA (0.0105
U) in phosphate buffer (37 °C) at 636 nm (ex 560 nm).
Supporting Information Available: Procedures and ad-
ditional data for syntheses and analyses (HPLC, MS, NMR)
reported herein. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL801582W
the self-immolative PABA linker between the phenylacetyl
moiety (i.e., PGA-sensitive substrate) and the phenol-based
fluorophore (Figure 2A). However, to avoid the laborious and
time-consuming handling and purification of highly polar
synthetic intermediates, the disulfonated indole 3b was intro-
duced at the final stage of the synthesis through a K2CO3-
(16) Turro, N. J. Modern Molecular Photochemistry; University Science
Books: Sausalito, CA, 1991.
(17) Hypsochromic shift of absorption maximum (λmax ) 470 nm) was
also observed.
(18) For instance, by using one of the numerous prodrug strategies
developed for biologically active phosphates and recently reviewed by
Hecker and Erion (Hecker, S. J. J. Med. Chem. 2008, 51, 2328–2345).
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