On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies

Article abstract of DOI:10.1021/jm050459+

The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.

Full text of DOI:10.1021/jm050459+

J. Med. Chem. 2005, 48, 7847-7859
7847
On the Use of Nonfluorescent Dye Labeled Ligands in FRET-Based Receptor
Binding Studies
Chouaib Tahtaoui,‡,∇ Fabrice Guillier,§,| Philippe Klotz,†,‡ Jean-Luc Galzi, Marcel Hibert,‡ and Brigitte Ilien*,
Laboratoire de Pharmacochimie de la Communication Cellulaire, Faculte´ de Pharmacie, UMR CNRS/ULP 7081,
74 route du Rhin, BP 24, 67401 Illkirch, France, De´partement Re´cepteurs et Prote´ines Membranaires, CNRS UMR 7100,
IFR 85, Boulevard Se´bastien Brant, BP 10413, 67412 Illkirch, France, and Faust Pharmaceuticals S.A., Bioparc,
Boulevard Se´bastien Brant, 67400 Illkirch, France
Received May 15, 2005
The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-
acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not.
We report here on the design, synthesis, and characterization of two novel pirenzepine
derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent
compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-
fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-,
and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor,
determined using either FRET or classical radioligand binding conditions. We provide evidence
that these compounds behave as potent acceptors of energy from excited EGFP with quenching
efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine
derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed
in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding
assays.
Introduction
bimolecular binding events that can be followed in real
time through FRET. Indeed, several G-protein coupled
receptors, such as the tachykinin NK2,9-11 the chemok-
ine CXCR4,12 and the muscarinic M receptors with their
N-termini fused to enhanced green fluorescent protein
(EGFP)13,14 behave as efficient donors for their respec-
tive ligands, designed as fluorescent acceptors with
appropriate spectral properties. Specific ligand binding
is recorded (on living cells and in real time) as a simple
variation of EGFP fluorescence emission, giving access
(i) to the measurement of kinetic as well as equilibrium
parameters of the interaction,9-14 (ii) to the identifica-
tion of different interconvertible receptor conformational
and functional states,10,11 and (iii) to the study of
allosteric modulators.14 Finally, miniaturized and au-
tomated FRET-based binding assays allow reliable
detection of unlabeled competitors under drug screening
conditions.13
Recently, several publications reported on the use of
nonfluorescent acceptors or quenchers such as dabcyl,15-17
the so-called black hole quenchers BHQ1,17,18 BHQ2,
and QSY-717 or QSY-9,19 nucleotides17 and especially
guanosine,20,21 or gold nanocrystals.16,22
Many of these studies describe molecular beacons
(hairpin-shaped oligonucleotides labeled with a fluoro-
phore and a quencher at their extremities) as powerful
tools to set up quenching/dequenching hybridization
assays.15,16,18,21 In dual-labeled molecular beacons, the
fluorescent reporter and the quencher dye are in such
close proximity that they share electrons, transiently
forming a nonfluorescent complex that absorbs light
energy, which is then dissipated as heat. Thus, very
efficient quenching takes place through static
quenching,17,18,22 a mechanism distinct from dynamic
Fo¨rster resonance energy transfer.7 Note that static
High-sensitivity methods are increasingly important
in biology and medicine and are expected to produce
major advances in molecular diagnostics, therapeutics,
molecular biology and bioengineering. Fluorescence-
based techniques are especially appealing because of the
potential sensitivity and specificity that can be achieved
(see, for review, refs 1 and 2). Among them, fluorescence
resonance energy transfer (FRET), which is intrinsically
dependent upon and highly sensitive to proximity
changes, represents a powerful monitoring/reporting
mechanism for intra- and intermolecular binding events,
both in vitro and in vivo.2-6
FRET is defined as the nonradiative transfer of the
excited-state energy from a donor fluorophore to an
acceptor molecule.7,8 It occurs if the emission spectrum
of the donor overlaps the excitation spectrum of the
acceptor and FRET efficiency depends on donor-accep-
tor separation.7 Thus, any event that brings the two
partners closer to (or farther from) each other will lead
to a dramatic change in energy transfer and hence
fluorescence.
Receptor-ligand interactions, classically studied by
separative radioligand binding techniques, are typical
† In fond memory of Dr. Philippe Klotz, our dear friend, colleague,
and mentor who died untimely on June 9th, 2005.
* To whom correspondence should be addressed. Mailing address:
De´partement Re´cepteurs et Prote´ines Membranaires, CNRS UMR
7100, Ecole Supe´rieure de Biotechnologie de Strasbourg, Boulevard
Se´bastien Brant, BP 10413, 67412 Illkirch, France. Tel: 00 33 (0)3 90
24 47 38. Fax: 00 33 (0)3 90 24 48 29. E-mail: ilien@esbs.u-strasbg.fr.
‡ UMR CNRS/ULP 7081.
∇ Present adress: Arpida AG, Research & Development of Anti-
Infectives, Dammstrasse 36, CH-4142 Muenchenstein, Switzerland.
§ Faust Pharmaceuticals S.A.
| Present adress: Laboratoires Fournier S.A., 50 rue de Dijon, 21121
Daix, France.
CNRS UMR 7100.
10.1021/jm050459+ CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/05/2005

7848 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Tahtaoui et al.
Figure 1. Chemical structures of dyes, dye-pirenzepine derivatives, and fluorescent pirenzepine congeners. Patent blue VF (1)
and pinacyanol (2) are commercially available dyes. The pirenzepine-spacer derivative (3)14 was used for the synthesis of the
nonfluorescent pirenzepine derivatives Pat(17)PZ, 4, and Pin(16)PZ, 5. Bo(15)PZ and RR(17)PZ are, respectively, the bodipy
[558/568] and rhodamine red labeled pirenzepine derivatives described in ref 14. For all these pirenzepine derivatives, the value
in parentheses specifies the number of atoms (spacer length) separating the chromophore from the pharmacophore, PZ.
quenching efficiency does not depend on spectral overlap
between donor fluorescence emission and acceptor ab-
sorbance.17
easier to synthesize, using commercially available and
often cheaper precursors. Thus, fine structural tuning
to get molecules endowed with particular absorption
properties prone to quenching optimization should be
possible.
Because all of these properties might be beneficial to
further improvement of FRET-based receptor binding
assays, we decided to examine whether a nonfluorescent
ligand could be designed and would behave as an
efficient acceptor partner for EGFP, taken as the donor
fluorophore. To these ends, we selected the well-
established model of muscarinic M1 receptors (EGFP-
(∆17)hM1 chimera) in interaction with the antagonist
pirenzepine.13,14
Two dyes, patent blue VF (1) and pinacyanol (2),
displaying acceptor-like spectroscopic properties, were
coupled to pirenzepine (3) to provide Pat(17)PZ (4) and
Pin(16)PZ (5) probes, respectively (Figure 1). The bind-
ing properties of these nonfluorescent derivatives were
assessed through FRET and classical [3H]-QNB (Qui-
nuclidinyl Benzilate) competition experiments. Quench-
ing efficiencies were calculated, giving access to the
estimation of donor-acceptor separation. Results are
discussed in light of those previously obtained, under
similar conditions, using fluorescent bodipy (Bo(15)PZ)
or rhodamine red (RR(17)PZ) pirenzepine analogues.14
Finally, special attention was paid to the critical and
Conversely, probe systems that place the two labels
at a longer distance (20-100 Å range), that is neverthe-
less appropriate for FRET require a significant spectral
overlap between donor fluorescence emission and quench-
er absorption for efficient energy transfer to be ob-
served.17 Available examples for dynamic FRET, quench-
ing include the binding of QSY9-labeled cyclodextrine
to fluorescent maltose binding protein (MBP),19 the use
of a series of staggered dual-labeled oligodeoxyribo-
nucleotide hybrids to examine the dependency of quench-
ing efficiency upon donor-quencher separation,17 the
monitoring of retroviral proteases activity using dabcyl-
quenched fluorogenic substrates,23 and the study of
hairpin ribozyme kinetics through guanosine-specific
quenching of fluorescein-labeled substrates.20
The use of nonfluorescent dyes has the particular
advantage of eliminating the potential problem of ligand
background fluorescence (nonsensitized emission) re-
sulting from direct acceptor excitation. Furthermore, the
palette of available quencher molecules offers a great
structural diversity,24 as compared to organic fluores-
cent probes, allowing the choice of appropriate acceptors
over a wide range of maximal absorption wavelengths.
From a chemical point of view, quenchers might be also

Nonfluorescent Dye Labeled Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7849
Scheme 1. Synthesis of Functionalized Patent Blue VF, 8a
a Legend: (i) POCl3, rt, 3 days; (ii) methyl-6-aminohexanoate. HCl, DIEA, DMF, rt, 5 h, 24% (i + ii); (iii) LiOH, dioxane/water (1/1), rt,
1.5 h, 40%; (iv) succinimideOH, DCC, DMF/DME (1/1), 65 °C, 20h, 73%. Inset: 2D-NOESY contacts for compound 6 in a 9:1 CDCl3/
CD3OD mixture.
experimental evaluation of advantages predicted from
the use of dye-labeled, instead of fluorescent, derivatives
in FRET studies.
The ester function of compound 6 was then saponified
with LiOH to give the acid 7 in a 40% yield. To prepare
the coupling reaction with the pirenzepine derivative
3, the carboxylic acid function of 7 was then activated
as its succinimidyl ester 8 (73% yield).
Results and Discussion
Because there is no commercially available substi-
tuted pinacyanol, we developed a strategy that involves
a convergent synthesis through the coupling of the two
quinaldinium intermediate molecules 14 and 17 (Scheme
2). The first one (14) was obtained in four steps starting
from 4-nitroaniline 9 and crotonaldehyde 10, which
were condensed under acidic and biphasic conditions
(Doebner-Miller conditions)25 to give the quinaldine 11.
Reduction of the nitro group afforded the aniline deriva-
tive 12, which was coupled with adipic acid monomethyl
ester, activated as its acyl chloride. Quinaldine 13 was
thus formed in 87% yield. Pyridine nitrogen was then
quaternarized into the first intermediate, quinaldinium
ethiodide 14, using ethyl iodide. Compound 17 was
formed in a one-step solvent-free reaction by coupling
ethylquinaldinium iodide 15 with N,N′-diphenylforma-
midine 16 at 140 °C and was then isolated by recrys-
tallization from ethanol. Finally, condensation of com-
pounds 14 and 17 in acetic anhydride and sodium
acetate led to the unsymmetrical pinacyanol 18. The
reaction turned very quickly to dark blue, which is the
characteristic color of the cyanine 18. Final saponifica-
tion of the ester function provided the corresponding
carboxylic pinacyanol acid 19 in 79% yield.
Two novel donor (EGFP fluorophore)-acceptor (non-
fluorescent ligand dye conjugate) pairs have been de-
signed, characterized, and compared for their binding
properties and FRET efficiency to EGFP-hM1 musca-
rinic receptors.
Two dyes, patent blue VF (1) and pinacyanol (2), were
functionalized and coupled to the pirenzepine derivative
3
14 to give the final nonfluorescent compounds 4 and 5
(Figure 1). The structures of two close fluorescent
analogues, the bodipy (Bo(15)PZ) and the rhodamine red
(RR(17)PZ) pirenzepine derivatives,14 are also pre-
sented. For homogeneity purposes, the two pirenzepine
conjugates 4 and 5 will be, respectively, referred to as
Pat(17)PZ and Pin(16)PZ, the value in parentheses
defining the number of atoms (spacer) separating the
pirenzepine pharmacophore from the chromophore mol-
ecule.
Synthesis of Nonfluorescent Dye Labeled Piren-
zepine Derivatives. Commercially available patent
blue VF 1 was first functionalized with methyl-6-
aminohexanoate after activation of the para-sulfonic
acid group with phosphorus oxychloride (Scheme 1),
leading to the corresponding methyl ester 6. This
compound was obtained as a single isolated isomer (24%
yield). Its 2D-NOESY analysis showed contacts between
the R-protons of the methyl-6-aminohexanoate chain
and the H3 and H5 protons of the patent blue moiety
(Scheme 1 inset). No contacts were found between
R-protons and the other protons of the dye moiety, in
particular, the H9 and H10 protons. Altogether, these
data indicate that the isolated product 6 is the 4-isomer.
Each of the two functionalized dyes, 8 and 19, were
coupled with the pirenzepine molecule 3 via the amino
function of the PEG spacer (Scheme 3). The patent blue
VF probe Pat(17)PZ (4) was formed using classical
coupling conditions via succinimidyl ester 8, diisopro-
pylethylamine, and molecule 3. For the pinacyanol probe
Pin(16)PZ (5), peptidic coupling conditions through acid

7850 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Scheme 2. Synthesis of Functionalized Pinacyanol, 19a
Tahtaoui et al.
a Legend: (i) PhCH3, HCl-6 N, reflux, 2 h, 60%; (ii) SnCl2, HCl, 100 °C, 30 min, 65%; (iii) adipic acid monomethyl ester, (COCl)2,
ꢀDMF, THF, rt, 1 h; (iv), THF, pyridine, 12, rt, 2 h, 87% (iii + iv); (v) EtI, PhCH3, sealed tube, 80 °C, 36 h, 46%; (vi) 140 °C, 30 min, 42%;
(vii) AcONa, AcOAc, 100 °C, 30 min, then KI, 56%; (viii) K2CO3, MeOH/H2O/CH3CN (8/1/1), rt, 36 h, 78%.
Scheme 3. Synthesis of the Nonfluorescent Dye Labeled Pirenzepine Derivatives 4 and 5a
a Legend: (i) 8, DIPEA, CH3CN/DMF (4/1), 3 h, rt; (ii) 19, BOP, NMM, DMF, 12 h, rt.
19, BOP reagent, and amine 3 were preferred. Coupling
reactions were performed on micromole amounts of the
reagents (see Experimental Section) and purification
was performed by high-pressure liquid chromatography
(HPLC). The expected final products were characterized
by mass spectroscopy.
blue VF (1)26 and pinacyanol (2)27,28 dyes exhibit ex-
tremely low quantum yields for fluorescence: 3.7 × 10-4
and 10-3, respectively. Overall spectral properties (maxi-
mal absorption wavelength and molecular extinction co-
efficient) reported for commercial dyes 1 (636 nm; 85 000
M-1‚cm-1 24,29 and 2 (604 nm; 175 000 M-1‚cm-1 24,28
) in
)
Spectral Properties of Functionalized Dyes and
Corresponding Pirenzepine Derivatives. Free patent
MeOH and in water are a priori compatible with those
expected for an energy acceptor from excited EGFP.

Nonfluorescent Dye Labeled Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7851
Since FRET experiments are performed in “physiologi-
cal” N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
(HEPES) buffer using living cells expressing EGFP-
fused muscarinic hM1 receptors, we decided to examine
the spectroscopic characteristics of the functionalized
dyes alone (7 and 19) or coupled to pirenzepine (Pat-
(17)PZ, 4, and Pin(16)PZ, 5) both in MeOH and in
HEPES/bovine serum albumin (BSA) buffer.
As shown in Figure 2a, the patent blue derivative 7
and its corresponding pirenzepine conjugate 4 (Pat(17)-
PZ) display identical spectral properties. In HEPES/BSA
buffer, as compared to MeOH, a slight 7 nm shift in
their maximal absorption wavelength is observed, to-
gether with a small decrease in molecular extinction
coefficient (Table 1). A similar bathochromic shift (7.5
nm) has been found to occur for the native patent blue
VF dye 1 in the presence of RNA aptamers, together
with a large increase in fluorescence quantum yield
(0.034) most likely due to restricted rotational freedom
of the dye within a more viscous environment.26
Figure 2b shows that functionalized pinacyanol 19
and Pin(16)PZ 5 spectra in MeOH are nearly superim-
posable. Besides a major peak at 615 nm, they exhibit
a band at 570 nm and a small shoulder at 525 nm. Such
a broad absorption spectrum, made of three overlapping
bands centered at 599-607, 550-570, and 520-525 nm,
has also been reported for the native dye 2, either in
MeOH, EtOH, water, or Tris buffer.24,28,30-32 Interest-
ingly, in HEPES buffer supplemented with BSA (1 mg/
mL), the two pinacyanol derivatives display complex
blue-shifted absorption spectra with different species in
equilibrium (Figure 2b). Indeed, it may be seen in Table
1 that λmax values and associated molecular extinction
coefficients for the three main absorbing species that
were identified depend on the dilution factor of the stock
solutions (0.68 mM in pure DMSO) of pinacyanol 19,
and Pin(16)PZ 5, in the buffer. The reduction in the
absorption of the most red-shifted bands, together with
the growth of the most energetic band and the overall
decrease in absorption intensity of 19 and 5, is indica-
tive of strong solvatochromic effects (hypsochromy,
metachromasia, and hypochromy).33,34 Such findings are
reminiscent of the various and profound rearrange-
ments of pinacyanol spectral properties already de-
scribed in the literature. Besides its ability to dimerize
in solution (ref 31 and references therein), this cationic
cyanine dye 2 is known for its high sensitivity to
medium polarity and refractive index28 and for its
fluorescence quantum yield that increases up to a value
of 0.014 upon binding to dendrimers.28 It has also
proven to be a useful extrinsic probe molecule of
aggregation and conformation states of various proteins
such as amyloid peptides31 and ovalbumin.30 It is
noteworthy that spectral changes that are recorded here
for pinacyanol derivatives 5 and 19 are very similar to
those resulting from the interaction of the native dye 2
with amyloid peptides in a â conformation.31 One may
speculate that the presence of BSA in the buffer that
was used here might be involved in the spectral changes
that we observed. Indeed, this globular protein has a
tendency to aggregate in macromolecular assemblies,
which may evolve toward typical â-sheet structures.35
Figure 2. Normalized absorption spectra of dyes and of their
pirenzepine derivatives in MeOH and in HEPES/BSA buffer:
(a) absorption spectra of the functionalized patent blue VF dye
7 (0,9) and of its pirenzepine derivative Pat(17)PZ 4 (O,b)
recorded in MeOH (open symbols) and in HEPES/BSA buffer
(filled symbols); (b) absorption spectra of functionalized pina-
cyanol 19 (0,9) and of its pirenzepine conjugate Pin(16)PZ 5
(4,2) recorded in MeOH (open symbols) and in HEPES/BSA
buffer (filled symbols; 0.3% final DMSO concentration); (c)
superimposition in HEPES/BSA buffer of Pat(17)PZ (b) and
of Pin(16)PZ (2; 0.5% final DMSO concentration) absorbance
with EGFP fluorescence emission (thick line; excitation at 440
nm) with overlaps for normalized spectra shown as hatched
or shaded areas, respectively. Maximal absorption wavelengths
and corresponding molecular extinction coefficients for Pin-
(16)PZ were as follows: 534 nm (61 800 M-1‚cm-1), 559 nm
(58 900 M-1‚cm-1), and 616 nm (36 250 M-1‚cm-1).
pirenzepine dye derivatives with EGFP fluorescence
emission. Spectral overlap integrals (J values) were
calculated using absorption spectra obtained with three
sets of dilutions of Pat(17)PZ and of Pin(16)PZ in
HEPES/BSA buffer (0.15-0.8% DMSO, v/v). Table 1
indicates that, at least in the probe dilution range that
was studied here and corresponds to that used in FRET
Finally, Figure 2c allows us to appreciate the great
differences in spectral overlap of absorbance of both

7852 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Tahtaoui et al.
Table 1. Spectroscopic Parameters for Dyes and Their Pirenzepine Derivatives
absorption
MeOH
HEPES/BSA buffer
donor-acceptor properties
overlap integral
Fo¨rster radius
compound
λmax (nm)
E (M-1‚cm-1
)
λmax (nm)
E (M-1‚cm-1
)
J (cm3‚M-1, ×10-14
)
R0 (Å)
7
636
636
570
615
85 000
85 000
78 700
175 000
643
644
78 000
78 000
b
b
Pat(17)PZ, 4
3.5 ( 0.5
38.9 ( 0.8
19 a
515 (508)
557 (558)
608 (607)
541 (531)
562 (556)
617 (611)
69 500 (71 400)
41 400 (29 700)
26 000 (16 600)
61 100 (64 900)
64 000 (57 400)
43 000 (32 200)
b
b
Pin(16)PZ, 5 a
571
615
82 800
175 000
32.9 ( 1.5
56.7 ( 0.4
a Absorption parameters in HEPES/BSA buffer were estimated in the presence of an identical 0.3% final DMSO concentration (v/v).
Values in parentheses are for a 0.6% (19) or a 0.8% (Pin(16)PZ) content in DMSO. λmax values and molecular extinction coefficients are
reported for all species that were detected. The spectral overlap integral (J), as well as the Fo¨rster radius (R0), was calculated as reported
in the Experimental Section. Mean values ( standard deviation are from three independent determinations performed at various dilutions
of the dye-pirenzepine derivatives in HEPES/BSA buffer (final DMSO concentrations ranging from 0.15 to 0.8%). b Not determined.
Table 2. Binding Properties of Dye Derivatives at EGFP(∆17)hM1 Receptorsa
fluorescence resonance energy transfer
[3H]-QNB
binding
Ki, nM
compound
Kd, nM
amplitude, %
efficiency, Ec
distance R, Å
pirenzepineb
23.5
7
.10 000
.10 000
189 ( 40
(n)4)
19
Pat(17)PZ, 4
30.1 ( 4.8
(n)3)
36.9 ( 1.1
(n)4)
0.52 ( 0.02
(n)4)
38.5 ( 0.4
(n)4)
Pin(16)PZ, 5
29.4 ( 9.2
(n)7)
69.7
53.5
9.1 ( 0.7
(n)6)
35.2 ( 0.7
(n)6)
0.48 ( 0.01
(n)6)
57.4 ( 0.3
(n)6)
Bo(15)PZb
RR(17)PZb
14.2
18.7
34.1
48.5
0.48
0.68
50.3
45.4
a Mean values ( standard errors are given for n independent determinations. b Values for pirenzepine and for its fluorescent derivatives
14
Bo(15)PZ and RR(17)PZ are from similarly conducted binding experiments.
c Efficiency values E were calculated from maximal
fluorescence extinction amplitudes (determined at equilibrium with saturating ligand concentrations) and taken to estimate donor-
acceptor separation (R values) as reported in the Experimental Section.
experiments, the solvatochromic effects on Pin(16)PZ (5)
absorption properties have little influence on the de-
termination of a mean J parameter with small associ-
ated standard deviation. Theoretical donor-acceptor
distances (Fo¨rster radius; R0 value) for 50% FRET
efficiency were then estimated (Table 1) and pointed to
a favorable R0 value for Pin(16)PZ. Moreover, its
negligible intrinsic fluorescence properties, added to its
broad spectral overlap with donor emission, make probe
5 a good candidate for efficient quenching of EGFP
fluorescence. Indeed, such properties are displayed by
many efficient dark quenchers,17-19 but they may also
be responsible for inner filter effects.19 As Pin(16)PZ (5)
significantly absorbs in the maximal EGFP excitation
(460 nm) and emission (510 nm) wavelength windows,
this possibility has to be considered.
Binding Properties of the Nonfluorescent Lig-
ands at EGFP-Fused M1 Receptors. The interaction
of the pirenzepine derivatives with M1 receptors was
examined on HEK cells expressing the EGFP(∆17)hM1
fluorescent chimera.
First, classical radioligand competition experiments
were performed with [3H]-QNB to verify the ability of
the new compounds to interact with the muscarinic
receptor. As reported in Table 2, dye moieties 7 and 19
exhibit very poor binding affinities. In contrast, Pin-
(16)PZ displays a nanomolar Ki-value comparable to
that of the antagonist pirenzepine and a 6-fold higher
affinity than Pat(17)PZ for hM1 receptors. For compari-
son, Bo(15)PZ and RR(17)PZ (two closely related fluo-
rescent analogues of the dye derivatives presented here)
were found to compete in the 50 nM concentration range
with [3H]-QNB for binding to the receptor.14
Figure 3 illustrates, through real-time monitoring of
EGFP fluorescence emission at 510 nm, several proper-
ties of dye 7 and Pat(17)PZ binding to EGFP(∆17)hM1
receptors. When added at an identical 500 nM concen-
tration to the cell suspension (Figure 3a), the derivatized
dye 7 leaves the fluorescence signal unchanged, while
Pat(17)PZ induces a strong and time-dependent fluo-
rescence extinction (up to 38%) reaching equilibrium
within 20 min at 21 °C. The small linear drift in basal
fluorescence that may be seen with 7 (and also with Pat-
(17)PZ at equilibrium) most probably arises from slow
cell settling. Thus, interaction of Pat(17)PZ with the
EGFP-fused receptor is driven by its pirenzepine moiety,
while its patent blue tag does not bind to the receptor
per se. The reversible character of Pat(17)PZ binding
to EGFP-(∆17)hM1 receptors is illustrated (Figure 3b)
by the ability of atropine (at a saturating concentration)
to induce a complete recovery in fluorescence emission.
Several similar experiments, performed at various Pat-
(17)PZ concentrations, indicated that dissociation fol-
lowed a monoexponential process with a mean koff value
of 0.0008 s-1 at 21 °C. Figure 3c visualizes the extent
of fluorescence extinction that was achieved at various
Pat(17)PZ concentrations. As expected, apparent as-
sociation rates increase with ligand concentration,
together with the amplitudes of fluorescence extinction,
which, however, stabilize around 35% at 400 nM Pat-
(17)PZ. As an internal control, commercial bodipy
[558/568] pirenzepine or Bo(10)PZ (one of the most

Nonfluorescent Dye Labeled Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7853
Figure 3. Examination of Pat(17)PZ binding properties through real-time monitoring of EGFP emission at 510 nm. In panel a,
dye 7 alone (O) or Pat(17)PZ (solid line) is added (arrow) at an identical 500 nM concentration to the EGFP(∆17)hM1 cell suspension.
In panel b, Pat(17)PZ (150 nM) is added (1) to the cell suspension, and binding is allowed to reach equilibrium. Thereafter,
atropine (5 µM) is added to the incubation medium (2) to initiate the dissociation step. Best fit trace (white line) for a
monoexponential fluorescence recovery (koff ) 0.001 s-1) is shown. In panel c, increasing concentrations of Pat(17)PZ are added
to the EGFP(∆17)hM1 cell suspension at time 0. Recording durations varied according to equilibrium time. As an internal control,
association of the fluorescent pirenzepine derivative Bo(10)PZ at 200 nM was also monitored (‚‚‚). Best fits for an exponential
decay of fluorescence are superimposed on each experimental curve. In panel d, amplitudes for maximal fluorescence extinction,
either measured at end-point time values of experimental curves shown in Figure 3c (b) or calculated from exponential fitting of
the same traces (0), are plotted as a function of Pat(17)PZ concentration. Best fit to the empirical Hill equation derived for saturation
is drawn. Estimated parameters are maximal FRET amplitude ) 37.9% ( 1.8%; Kd ) 33.4 ( 2.7 nM; nH ) 1.20 ( 0.11.
potent fluorescent acceptors that we have previously
described)13,14 led at a saturating 200 nM concentration
to an expected 46% decrease in fluorescence emission.
Association traces shown in Figure 3c were superim-
posed with their best exponential fits to derive EGFP
fluorescence extinction amplitude values at infinite incub-
ation time. These FRET amplitudes, together with those
determined experimentally, were then plotted as a func-
tion of Pat(17)PZ concentration (Figure 3d). Both deter-
minations were equivalent, indicating that equilibrium
was reached at all ligand concentrations. A typical
saturation curve was generated, allowing the determi-
nation of several parameters such as a maximal FRET
amplitude of 37.9% ( 1.8% at saturation, a Kd value of
33.4 ( 2.7 nM and a Hill coefficient of 1.2 ( 0.1.
The second pinacyanol series of ligands was then
investigated under similar FRET-based binding condi-
tions. In contrast with the former patent blue VF
derivatives, functionalized pinacyanol 19 and Pin(16)-
PZ both promoted an immediate and concentration-
dependent drop in fluorescence upon addition to the cell
suspension. This rapid event was followed, exclusively
in the presence of Pin(16)PZ (as illustrated in Figure
4a, step 1), by a slower time-dependent extinction
process that reached equilibrium. Upon addition of 5
µM atropine (step 2), only the slow association compo-
nent of Pin(16)PZ was abolished with a koff of 0.0004
s-1 for fluorescence recovery at 21 °C. Accordingly, the
sudden decrease of EGFP fluorescence, which was
observed using Pin(16)PZ (and the inactive derivative
19, not shown), was not prevented by preincubating the
cells with a saturating atropine concentration (step 3)
and was thus clearly due to a nonspecific phenomenon.
This artifact probably stems from a concentration-
dependent inner filter effect of the pinacyanol dyes,
which were shown to strongly absorb (Figure 2c) in the
excitation/emission wavelength window (460-510 nm)
of EGFP.13 In all subsequent experiments, specific Pin-
(16)PZ binding was defined as the slow-associating
component and more accurately as the amplitude of
fluorescence extinction that was sensitive to atropine.
Initial fluorescence levels (100%) were thus set, for each
probe concentration, as remaining cell fluorescence with
fully atropine-occupied receptors (Figure 4a; dashed
line). The robustness of this definition is illustrated in
Figure 4a both by prevention and dissociation experi-
ments, which indeed led to the determination of an
identical FRET amplitude (33.4%) for Pin(16)PZ binding
at 100 nM. Figure 4b presents the results of six
independent saturation experiments and shows that
reliable determinations of Kd values (9.1 ( 0.7 nM; nH
) 1.10 ( 0.08) and of maximal FRET amplitudes (35.2%
( 0.7%) for Pin(16)PZ binding to hM1 receptors were
obtained.

7854 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Tahtaoui et al.
Estimation of Donor-Acceptor Separation. Be-
cause Pat(17)PZ (4) and Pin(16)PZ (5) probes display
efficiency values for EGFP donor extinction close to 50%,
calculation of donor-acceptor separation led to R values
close to the actual Fo¨rster radii (R0 values) listed in
Table 1. Former comparison of a series of bodipy-
pirenzepine derivatives, differing by the nature and the
length of the spacer introduced between pirenzepine and
the fluorophore, led us to propose the existence of a
receptor subsite where the bodipy group is locked at a
45-50 Å distance from the EGFP fluorophore.14
Here, the distance separating EGFP from receptor-
bound pinacyanol in Pin(16)PZ is much more difficult
to ascertain. Indeed, this dye displays complex spectral
properties, which are very sensitive to environmental
changes and which probably depend on the free or
bound state of the probe.28,30,31 Such features make the
determination of molecular extinction coefficient and R0
values more uncertain. Because inner filter effects may
also interfere with the quantification of a specific EGFP
extinction signal, it is clear that the R value (57.4 Å)
presented here for Pin(16)PZ can only be regarded as a
rough estimate of the actual donor-acceptor distance.
It remains that all these problems are not encoun-
tered with Pat(17)PZ. When bound to hM1 receptors,
its chromophore is apparently about 7-11 Å closer to
EGFP than the bodipy or rhodamine red fluorophores.14
One may suggest that patent blue VF, due to its overall
hydrophilicity, escapes from a putative fluorophore
receptor binding pocket and protrudes out of the trans-
membrane core of the receptor toward the extracellular
medium and EGFP.
On the Use of Fluorescent versus Nonfluores-
cent FRET Acceptors. In the context of FRET-based
receptor binding assays, several advantages are ex-
pected for quencher-type ligands over fluorescent probes.
Of course, this preferentially applies to dye derivatives,
such as Pat(17)PZ, exhibiting appropriate spectral
properties and negligible absorbance at donor excitation
wavelength.
A main improvement in the reliability of donor
extinction measurements (FRET signal) is expected
from the abolition of interfering background acceptor
emission, often detectable at micromolar concentrations
of fluorescent ligands. This crucial point has been
checked (Figure 5) by comparing Bo(10)PZ and Pat(17)-
PZ binding properties at high concentrations. Figure 5a
shows that binding of the dye-labeled pirenzepine
derivative can be monitored within a concentration
range reaching up to 500-fold its Kd value (Figure 3c,d).
As expected, association kinetic velocities increase as a
function of ligand concentration. Most importantly,
amplitudes for EGFP fluorescence extinction at equi-
librium are not affected at high dye concentrations
(Figure 5a insert) and remain in full agreement with
the mean value for maximal FRET amplitude reported
for this compound in Table 2. Fluorescence emission
spectra recorded on cell suspensions preincubated or not
with high Pat(17)PZ concentrations indicate too that
maximal emission wavelength for EGFP (508 nm) was
not shifted upon ligand addition (not shown). Figure 5b
depicts a parallel experiment conducted with Bo(10)-
PZ, which highlights major drawbacks that are associ-
ated to the use of a fluorescent probe at high concen-
Figure 4. Assessment of receptor specificity for Pin(16)PZ-
mediated cell fluorescence extinction. In panel a, fluorescence
emission (excitation set at 470 nm) of EGFP(∆17)hM1 cells
was monitored in real time following the addition of 100 nM
Pin(16)PZ (step 1). Subsequent addition of 5 µM atropine (re-
ferred to time 0 of dissociation) led to a partial recovery in
initial fluorescence with a koff value of 0.004 s-1 (step 2). In a
separate experiment (0), 100 nM Pin(16)PZ was added to cells
preincubated with a saturating 5 µM atropine concentration
(step 3). Specific FRET amplitude (%) was calculated as the
fractional atropine-sensitive extinction of control (dashed line)
fluorescence levels. For this typical experiment, a specific
FRET amplitude of 33.4% was determined. In panel b, a typical
occupancy curve for Pin(16)PZ binding to EGFP(∆17)hM1 re-
ceptors was generated by reporting specific FRET amplitudes
(mean values with associated standard error bars), which were
obtained at various Pin(16)PZ concentrations from six inde-
pendent experiments. Best fit to the empirical Hill equation
derived for saturation is drawn (maximal FRET amplitude )
35.2% ( 0.7%; Kd ) 9.1 ( 0.7 nM; nH ) 1.10 ( 0.08).
Table 2 summarizes the binding properties of the four
nonfluorescent derivatives, together with those of two
close fluorescent congeners (Bo(15)PZ and RR(17)PZ).14
First, whatever the way used to determine their binding
affinity constants, the four pirenzepine derivatives
interact with nanomolar affinity with the EGFP(∆17)-
hM1 fluorescent chimera, Pin(16)PZ being the most
potent among them. As previously discussed, the con-
stant tendency of compounds to exhibit higher affinities
when tested by FRET may rely on differences in
experimental conditions.14 Second, it may be observed
that maximal FRET amplitudes achieved at saturating
concentrations of the two nonfluorescent pirenzepine
derivatives and of Bo(15)PZ are similar, thereby the
corresponding efficiency values E. Note, however, that
these values did not reach the efficiency afforded with
RR(17)PZ, the most sensitive fluorescent acceptor that
we previously selected.14

Nonfluorescent Dye Labeled Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7855
(EGFP fluorescence extinction) with the time course of
the strong Bo(10)PZ emission interference, which sta-
bilizes upon ligand homogenization into the incubation
medium, and finally (iii) markedly reduced fluorescence
extinction amplitudes that represent less than 6% (at
4.5 µM Bo(10)PZ) of initial cell fluorescence in the
absence of ligand. Thus, at high Bo(10)PZ concentra-
tions, direct monitoring of fluorescence emission at 510
nm no longer reflects solely specific ligand binding to
EGFP(∆17)hM1 receptors. Instead, it represents a
mixed contribution of both a decrease in EGFP emission
(through FRET) and an increase in basal fluorescence
levels due to intrinsic ligand emission (with excitation
set at 470 nm). This explanation is supported by the
findings (Figure 5b) that the addition of atropine (10
µM) to cells equilibrated in the presence of 4.5 µM Bo-
(10)PZ induces a slow recovery in overall fluorescence
that reaches a plateau at 145% of initial cell fluores-
cence at 510 nm. The amplitude (49%) of this monoex-
ponential process is close to that found for EGFP
extinction using Bo(10)PZ at 200 nM, and its rate
constant (0.0003 s-1) is comparable to the off-rate
constant (0.0005 s-1) previously defined14 for Bo(10)PZ
dissociation from EGFP(∆17)hM1 receptors.
Altogether, these results highlight the superiority of
Pat(17)PZ (a derivative that exhibits also negligible ab-
sorbance at 470 nm, i.e., the donor excitation wave-
length) over Bo(10)PZ for performing safe FRET-based
binding measurements requiring micromolar concentra-
tions of ligand. As a direct consequence, such nonfluores-
cent derivatives should help in expanding the number
and variety of “FRET-targeted” receptors, including those
displaying low-affinity ligand-receptor interactions.
Another strong argument for the use of nonfluorescent
versus fluorescent dyes is connected to the environmen-
tal dependency of fluorescence quantum yields8 that
may greatly differ for free or specifically or nonspecifi-
cally bound probes. Thus, an increase in quantum yield
for an acceptor fluorophore could lead to an elevation
in its background emission interference. Despite the
increase in quantum yield reported for dyes 1 (0.034)
and 2 (0.014) when interacting with RNA aptamers26
and host dendrimers,28 respectively, such a phenomenon
is unlikely to occur with these dyes as their quantum
yield values still remain very low.
Though intensity-based FRET detection methods
include monitoring the donor intensity, the sensitized
acceptor emission, or the ratio between donor and accep-
tor intensity, it has been found that the fraction of sen-
sitized acceptor emission is hardly detectable in binding
assays usually performed at large ligand over receptor
molar excess.9,13 In such a context, dye acceptors may
be regarded as equivalent to fluorescent ones because
donor emission is the unique parameter that allows
reliable and sensitive detection of a FRET process.
Importantly too, nonfluorescent molecules are not sub-
jected to photobleaching, a phenomenon characterized
by an irreversible loss of fluorescence properties due to
photon-induced chemical damage and covalent modifi-
cation. Finally and from a more practical point of view,
dyes, which are cheaper and probably more stable than
fluorophores, may be regarded as interesting tools for
high-throughput FRET-based screening methods.
Figure 5. Real-time monitoring of fluorescence emission at
510 nm upon addition of micromolar concentrations of Pat-
(17)PZ and Bo(10)PZ to EGFP(∆17)hM1 expressing cells.
Fluorescence at 510 nm (excitation set at 470 nm) was recorded
in real time and normalized to 100% for initial cell fluorescence
in the absence of ligand. Aliquots of a concentrated stock
DMSO solution of Pat(17)PZ (a) or of Bo(10)PZ (b) were added
at 50 s (arrow) to the cell suspension to give the final indicated
concentrations. In the inset in panel a, amplitudes for fluo-
rescence extinction at binding equilibrium of Pat(17)PZ at
various concentrations are given. In panel b, variations in
fluorescence emission intensity due to the addition to the cell
suspension of Bo(10)PZ at 0.2 µM (control), 1.5 µM (0), or 4.5
µM (2) are shown. Once the fluorescence signal in the presence
of 4.5 µM Bo(10)PZ was stabilized (around 420 s), atropine
(10 µM) was added (time 0; top x-axis) to the incubation
medium, and fluorescence recovery was recorded with time.
The resulting dissociation trace is superimposed with its
monoexponential fit (4).
tration. At a 200 nM saturating concentration, Bo(10)PZ
binding proceeds as expected for a control experiment
with a FRET amplitude at equilibrium (44.2%) similar
to that obtained in Figure 3c (46%) or previously
reported for the same receptor construct (45%).13,14 In
contrast, further increases in the concentration of the
fluorescent ligand (up to 45 times the concentration
needed to occupy all receptor sites or up to 350-fold its
Kd value13,14) enable several observations to be made:
(i) marked transient injection artifacts due to interfering
ligand emission (at 510 nm) at the time of its addition
(aliquot of a concentrated stock solution in DMSO) to
the cell suspension, (ii) altered kinetics that do not
follow mass action and probably result from the super-
imposition of true ligand binding kinetics to the receptor

7856 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Tahtaoui et al.
coupling constants (J) are measured in hertz. The signals are
described as s (singlet), d (doublet), t (triplet), q (quadruplet),
m (multiplet), and br s (broad singlet). Electrospray ionization
(ESI)-MS were recorded on a Perkin-Elmer Biospec mass
spectrometer. MALDI-TOF measurements were carried out on
a Brucker BIFLEX spectrometer equipped with the SCOUT
high-resolution optics. Silica gel 60 (63-200 µm) from Merck
was used for column chromatography. The melting points are
measured in open capillary tubes using a Gallekampf ap-
paratus and are uncorrected.
Conclusion
This work demonstrates that ligand-receptor inter-
actions of muscarinic M1 receptors can be monitored
and characterized through FRET using a fluorescent
donor (EGFP) and a nonfluorescent acceptor. Though a
variety of quenchers for various synthetic fluorophores
have already been described, 16-18,21 we provide evidence
here that dyes, which constitute a wide family of
chemicals, may represent appropriate energy acceptors
from excited EGFP and possibly also from other GFP
variants.
Because donor and acceptor moieties are present on
separate molecules, able to form intermolecular recep-
tor-ligand complexes, EGFP extinction most likely
relies here on a dynamic Fo¨rster mechanism.8,17,18 This
is at variance with most quenchers described in the
literature that have been found to act intramolecularly,
through a static or contact mode of quenching.17,18,22
Pinacyanol and patent blue derivatives of pirenzepine
are endowed with high affinity for the muscarinic M1
receptor and quench EGFP fluorescence with efficiencies
comparable to those previously reached using fluores-
cent pirenzepine derivatives.14 With the example of Pin-
(16)PZ, we show that quenchers that exhibit broad
absorption spectra might also lead to undesirable inner
filter effects,19 a problem we circumvented by careful
assessment of FRET signal specificity, that is, by
monitoring an atropine-sensitive variation in donor
emission.
Clearly, Pat(17)PZ represents a more suitable com-
pound to get straightforward, reliable, and sensitive
measurements of ligand-receptor interactions through
FRET. Furthermore the synthesis of other derivatives,
displaying greater spectral overlap with donor emission
together with minimal absorption at donor excitation
wavelength, should help in improving the sensitivity of
the method and expanding the panel of “FRET-targeted”
receptors. In contrast to binding techniques that make
use of a single labeled molecule (either radioactive or
fluorescent) and thus require high-affinity ligands, the
development of FRET assays using nonfluorescent dye
derivatives is expected to lead to broad applications in
the receptor field. Indeed, it should give access to the
study of low-affinity ligand-receptor interactions (which
require micromolar concentrations of probe) while pre-
serving the great receptor specificity of the method
arising from donor (EGFP fused to the receptor of
interest) extinction measurement. These are very prom-
ising conditions that open, for example, the possibility
to start the characterization of orphan receptors through
the selection of tracer candidates from dye-labeled
chemical libraries.
Methyl 6-(4-[(4-Diethylaminophenyl)(4-diethylimino-
2,5-cyclohexadien-1-ylidene) methyl]-3-sulfo-1-phenyl-
sulfonamido)hexanoate (6). Phosphorus oxychloride (40
mL) was added to patent blue VF, 1 (50%, 5.66 g, 5.2 mmol),
placed in a 100 mL round-bottom flask. The mixture was
vigorously stirred at room temperature for 3 days before
phosphorus oxychloride was removed under reduced pressure.
Toluene was added to the mixture, and POCl3 was removed
by azeotropical distillation. The crude oil was then dissolved
in DMF (10 mL), and this solution was added dropwise to a
solution of methyl-6-aminohexanoate hydrochloride (8.0 g, 44
mmol) and DIPEA (6.45 g, 50 mmol) in DMF (40 mL). The
reaction mixture was stirred at room temperature for 5 h, and
DMF was evaporated under reduced pressure. The crude
resulting product was then taken up in CH2Cl2 and washed
with water. After separation, the organic layer was concen-
trated and purified by silica gel column chromatography to
give the methyl ester derivative 6 as blue crystals after
trituration in Et2O (0.82 g, 24%). TLC (CH2Cl2/MeOH 9/1) Rf
0.6; mp >250 °C; 1H NMR (CDCl3-10%CD3OD, 300 MHz) δ
8.55 (d, J ) 1.5 Hz, 1H), 7.87 (dd, J1 ) 1.5 Hz, J2 ) 7.5 Hz,
1H), 7.37 (d, J ) 9.5 Hz, 4H), 7.11 (d, J ) 7.5 Hz, 1H), 6.74 (d,
J ) 9.5 Hz, 4H), 3.62 (s, 3H), 3.54 (qd, J ) 7 Hz, 8H), 2.93
(qd, J ) 8 Hz, 2H), 2.28 (t, J ) 7.5 Hz, 2H), 1.58-1.52 (m,
4H), 1.34 (m, 2H), 1.26 (t, J ) 7 Hz, 12H); ESI-TOF (90 eV)
m/z calculated 672.28 (C34H46N3O7S2), found 672.21 (M)+.
{4-[[4-(5-Carboxy-pentylsulfamoyl)-2-sulfo-phenyl]-(4-
diethylamino-phenyl)-methylene]-cyclohexa-2,5-die-
nylidene}-diethylammonium (7). A solution of lithium
hydroxide hydrate (34 mg, 0.81 mmol) in water (10 mL) was
added dropwise at room temperature to the methyl ester
derivative 6 (170 mg, 0.25 mmol) dissolved in dioxane (10 mL).
The reaction was stopped after 1.5 h and before the total
conversion of the reaction, because TLC monitoring indicated
the formation of a polar side product. The dioxane solvent was
removed under reduced pressure, and water (10 mL) was
added. The mixture was washed with CH2Cl2 (4 times),
acidified with a 1 M KHSO4 solution, and extracted with CH2-
Cl2 (5 times) until the aqueous layer became pale red. After
concentration of the CH2Cl2 layer (without drying over mag-
nesium sulfate), the crude product was purified by silica gel
column chromatography to give the carboxylic acid derivative
7 as a blue solid (67 mg, 40%). TLC (CH2Cl2/MeOH 9/1), Rf
0.25; mp >250 °C; 1H NMR (CD3OD,200 MHz.) δ 8.86 (s, 1H),
8.21 (d, J ) 8 Hz, 1H), 7.66 (d, J ) 9 Hz; 4H), 7.50 (d, J ) 8
Hz, 1H), 7.11 (d, J ) 9.4 Hz, 4H), 3.88 (qd, J ) 7.2 Hz, 8H),
3.25 (t, J ) 6.9 Hz, 2H), 2.53 (t, J ) 7.2 Hz, 2H), 1.85-1.76
(m, 4H), 1.65 (m, 2H), 1.58 (t, J ) 7.2 Hz, 12H).
[4-((4-Diethylamino-phenyl)-{4-[5-(2,5-dioxo-pyrrolidin-
1-yloxycarbonyl)-pentylsulfamoyl]-2-sulfo-phenyl}-me-
thylene)-cyclohexa-2,5-dienylidene]-diethylammoni-
um (8). A mixture of N-hydroxysuccinimide (26 mg, 0.226
mmol) and DCC (46 mg, 0.223 mmol) dissolved in DME (5 mL)
was added at room temperature onto a solution of the car-
boxylic acid derivative 7 (65 mg, 0.1 mmol) dissolved in DMF
(5 mL). The mixture was stirred at 65 °C for 20 h, and solvents
were then removed under reduced pressure. The crude residual
product was taken up in CH2Cl2 and washed with water. After
concentration of the CH2Cl2 layer (without drying), the crude
product was finally boiled with AcOEt for 5 min and filtered
to remove the solubilized DCU. Upon filtration, the succinim-
idyl ester 8 was recovered as blue crystals, which were washed
Experimental Section
General Methods. All chemicals were obtained from
commercial suppliers and used without further purification.
Patent blue VF [129-17-9] was obtained from ACROS with
a 50% dye content. Pyridine was dried over KOH, and DMF
was dried on P2O5 and distilled from KOH at 70 °C under
reduced pressure. Tetrahydrofuran (THF) was distilled from
sodium-benzophenone. Acetonitrile (CH3CN) was distilled from
CaH2. NMR spectra were recorded on a Brucker DPX spec-
trometer at 200 and 300 MHz (1H), at 50 and 75 MHz (13C),
and at 200 MHz (2D-NOESY). NMR chemical shifts are
expressed in ppm relative to internal solvent peaks, and
1
with hot AcOEt (55 mg, 73%). Mp >250 °C; H NMR (CDCl3
200 MHz) δ 8.51 (s, 1H), 7.82 (d, J ) 7.1 Hz, 1H), 7.31 (d, J )

Nonfluorescent Dye Labeled Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7857
9.0 Hz, 4H), 7.07 (d, J ) 8.1 Hz, 1H), 6.69 (d, J ) 8.8 Hz, 4H),
3.47 (qd, J ) 6.8 Hz, 8H), 2.89 (t, 2H), 2.75 (s, 4H), 2.52 (t, J
) 6.1 Hz, 2H), 1.66 (t, J ) 6.1 Hz, 2H), 1.50-1.35 (m, 4H),
1.23 (t, J ) 6.6 Hz, 12H).
stirred under argon at 140 °C during 30 min. The obtained
brownish solid was then recrystallized from HCl, 2 N. The
yellow solid 17 was then filtered off and dried under vacuum
(280 mg, 42%). 1H NMR (DMSO, 200 MHz) δ 11.62 (br s, 1H),
8.97 (d, J ) 11.7 Hz, 1H), 8.38 (q, J ) 8.5 Hz, 2H), 8.12 (d, J
) 9.1 Hz, 1H), 8.02 (d, J ) 8.1 Hz, 1H), 7.89 (t, J ) 7.8 Hz,
1H), 7.62 (t, J ) 7.34 Hz, 1H), 7.38-7.52 (m, 4H), 7.15 (t, J )
7.1 Hz, 1H), 6.45 (d, J ) 11.7 Hz, 1H), 4.57 (q, J ) 6.8 Hz,
2H), 1.48 (t, J ) 7.1 Hz, 3H); 13C NMR (DMSO, 50 MHz) δ
155.3, 149.4, 139.5, 138.4, 137.9, 133.1, 129.7, 129.6, 125.1,
124.4, 119.0, 117.1, 116.9, 94.2, 44.3, 12.4; ESI-TOF (90 eV)
m/z calculated 402.06 (C19H19IN2), found 275.15 (M - I)+.
1-Ethyl-2-[(1E,3E)-3-(1-ethylquinolin-2(1H)-ylidene)
Prop-1-enyl-6-[(6-methoxy-6-oxo-hexanoyl) amino] Quin-
olinium Iodide (18). A mixture of compound 14 (70 mg, 0.15
mmol), compound 17 (68 mg, 0.17 mmol), and anhydrous
sodium acetate (25 mg, 0.31 mmol) in acetic anhydride (5 mL)
was heated at 100 °C during 30 min. The mixture changes
color rapidly to dark blue, which is the characteristic color of
the cyanine dye. The final compound 18 was then precipitated
by addition of a saturated aqueous solution of KI. The solid
was filtered off, washed with water, and dried under reduced
pressure (55 mg, 56%). 1H NMR (DMSO, 300 MHz) δ 9.80 (br
s, 1H), 8.56 (t, J ) 11.3 Hz, 1H), 7.40-8.15 (m, 11H), 6.46-
6.59 (m, 2H), 4.50 (q, J ) 9 Hz, 4H), 3.63 (s, 3H), 2.38 (m,
4H), 0.88-1.70 (m, 10H); 13C NMR (DMSO, 75 MHz) δ 172.4,
170.8, 150.9, 146.2, 138.3, 135.9, 135.3, 134.3, 131.6, 128.4,
124.2, 124.0, 123.8, 119.8, 119.6, 117.0, 116,2, 115.1, 105.1,
104.0, 50.4, 42.5, 42.0, 35.5, 32.6, 23.9, 23.5, 12.0, 11.7; ESI-
TOF (90 eV) m/z calculated 637.18 (C32H36IN3O3), found 510.25
(M - I)+.
6-[(5-Carboxypentanoyl) amino]-1-ethyl-2-[(1E,3E)-3-
(1-ethylquinolin-2 (1H)-ylidene) prop-1-enyl] Quinolin-
ium Iodide (19). Compound 18 (50 mg, 0.0785 mmol) was
dissolved in a mixture of MeOH/H2O/CH3CN (8/1/1, 10 mL),
and K2CO3 (22 mg, 0.157 mmol) was added. The reaction was
stirred during 36 h at room temperature. The reaction mixture
was then evaporated to dryness under reduced pressure, and
water was added (10 mL). The pH of the obtained solution was
then rectified to 4-5 by addition of HCl, 1 N. The obtained
precipitate was filtered off and dried under high vacuum to
give compound 19 as a blue solid (38 mg, 78%). 1H NMR
(DMSO, 200 MHz) δ 12.03 (br s, 1H), 10.23 (br s, 1H), 8.58 (t,
J ) 11.9 Hz, 1H), 8.22 (t, J ) 11.7 Hz, 2H), 7.67-8.00 (m,
7H), 7.36 (t, J ) 7.2 Hz, 1H), 6.56 (d, J ) 12 Hz, 1H), 6.45 (d,
J ) 11.5 Hz, 1H), 4.40 (q, J ) 7.3 Hz, 4H), 2.25-2.35 (m, 4H),
1.01-1.60 (m, 10H); 13C NMR (DMSO, 75 MHz) δ 174.3, 171.4,
151.2, 151.1, 146.7, 138.6, 136.3, 135.8, 134.8, 134.3, 131.1,
128.9, 125.2, 124.4, 124.2, 120.3, 120.0, 126.9, 125.7, 105.6,
104.5, 43.0, 42.6, 36.1, 33.4, 24.6, 24.1, 12.7, 12.4.
Preparation of the Nonfluorescent Dye Labeled Piren-
zepine Derivatives 4 and 5. The dye-labeled derivatives
were obtained by coupling the spacer-linked pirenzepine 3
dissolved in dry DMF with the corresponding dye at room
temperature (Scheme 3). The reaction was monitored by HPLC
and stopped upon complete disappearance of the dye reagent
(after 3 h for compound 8 (probe 4) and 12 h for compound 19
(probe 5)). The products were purified under reversed-phase
HPLC conditions (as detailed below) and eluted at retention
time tr. The fractions of interest were pooled, concentrated in
a Speed Vac concentrator (ISS 100 Savant), and further
checked for purity by analytical HPLC (using different elution
conditions as reported below). They were all found to elute as
single symmetrical peaks at retention time tr. The identities
of the compounds were analyzed by MALDI-TOF or ESI-TOF.
2-Methyl-6-nitro-quinoline (11). 4-Nitroaniline (616 mg,
4.46 mmol) was dissolved in HCl, 6 N (23 mL), and the mixture
was heated at 100 °C. Crotonaldehyde (0.74 mL, 8.92 mmol)
in toluene (6 mL) was added dropwise, and the reaction was
heated 2 h at 100 °C, then cooled to room temperature and
basified with KOH until pH 10-11. The mixture was extracted
twice with CH2Cl2, and the organic layer was washed with
brine and dried over MgSO4. The final quinaldine 11 was
obtained after purification by column chromatography on silica
gel (503 mg, 60%). TLC (CH2Cl2), Rf 0.15; 1H NMR (CDCl3,
200 MHz) δ 8.72 (d, J ) 2.7 Hz, 1H), 8.42 (dd, J1 ) 9.3 Hz, J2
) 2.7 Hz, 1H), 8.20 (d, J ) 8.54 Hz, 1H), 8.10 (d, J ) 9.3 Hz,
1H), 7.44 (d, J ) 8.6 Hz, 1H), 2.79 (s, 3H); 13C NMR (CDCl3,
50 MHz) δ 163.3, 153.6, 150.0, 137.6, 130.4, 125.2, 124.3, 123.9,
122.9, 25.7; ESI-TOF (90 eV) m/z calculated 188.06 (C10H8N2O2),
found 189.05 (M + H)+.
2-Methyl-quinolin-6-ylamine (12). To a solution of com-
pound 11 (500 mg, 2.66 mmol) in HCl, 1 N (20 mL), a solution
of SnCl2 (2.52 g, 13.3 mmol) in HCl, 1 N (10 mL), was added.
The mixture was heated at 100 °C during 15 min and then
cooled to room temperature. The precipitate was filtered,
washed with a solution of KOH (1 N) until pH 11-12, and
extracted with AcOEt. After removal of the solvent under
reduced pressure, the expected compound was purified by silica
gel column chromatography to afford 12 as a white solid (274
1
mg, 65%). TLC (AcOEt), Rf 0.28; H NMR (CDCl3, 200 MHz)
δ 7.81 (d, J ) 8.8 Hz, 1H), 7.77 (d, J ) 7.6 Hz, 1H), 7.12 (t, J
) 6.6 Hz, 2H), 6.84 (d, J ) 1.7 Hz, 1H), 3.88 (br s, 2H), 2.65
(s, 3H); 13C NMR (CDCl3, 50 MHz) δ 155.1, 143.9, 142.9, 134.1,
129.7, 127.8, 122.2, 121.3, 107.7, 24.9.
Methyl 6-[(2-Methylquinolin-6-yl) amino]-6-oxohex-
anoate (13). To a solution of the adipic acid monomethyl ester
(324 mg, 2.02 mmol) in THF (10 mL), oxalyl chloride (0.175
mL, 2.02 mmol) was added dropwise. A few drops of DMF (0.1
mL) was added to the mixture. Stirring was continued 1 h at
room temperature; then the reaction mixture was evaporated
to dryness. The crude product was dissolved in THF (10 mL);
then pyridine was added (0.615 mL, 7.6 mmol), followed by
the compound 12 (200 mg, 1.26 mmol). The reaction mixture
was stirred 2 h at room temperature, then washed with HCl,
0.5 N, and extracted with AcOEt. The organic layers were
washed with brine, dried over MgSO4, filtered, and evaporated.
The final product was obtained after purification by column
chromatography on silica gel (331 mg, 87%). TLC (CH2Cl2/
AcOEt 5/5). Rf 0.30; 1H NMR (CDCl3, 200 MHz) δ 8.35 (d, J )
1.96 Hz, 1H), 7.99 (t, J ) 8.55 Hz, 2H), 7.76 (br s, 1H), 7.54
(dd, J1 ) 9.04 Hz, J2 ) 2.44 Hz, 1H), 7.27 (d, J ) 8.54 Hz,
1H), 3.70 (s, 3H), 2.73 (s, 3H), 2.37-2.49 (m, 4H), 1.74-1.82
(m, 4H); 13C NMR (CDCl3, 50 MHz) δ 174.1, 171.3, 157.8, 144.6,
136.3, 135.4, 128.7, 127.0, 125.9, 123.2, 122.6, 116.1, 51.6, 37.1,
33.6, 24.8, 24.2; ESI-TOF (90 eV) m/z calculated 300.14
(C17H20N2O3), found (M + H)+ 301.13.
1-Ethyl-6-[(6-methoxy-6-oxohexanoyl) amino]-2-meth-
ylquinolinium Iodide (14). Compound 13 (100 mg, 0.33
mmol) dissolved in anhydrous toluene (3 mL) with ethyl iodide
(54 µL, 0.66 mmol) was heated in a sealed tube at 80 °C during
36 h. The reaction mixture was evaporated to dryness under
reduced pressure, and the crude product was triturated in
AcOEt. The precipitate was filtered off, washed several times
with AcOEt, and dried under high vacuum to give compound
14 as a pinkish solid (70 mg, 46%). 1H NMR (DMSO, 200 MHz)
δ 10.82 (br s, 1H), 9.03 (d, J ) 8.3 Hz, 1H), 8.77 (s, 1H), 8.62
(d, J ) 9.52 Hz, 1H), 8.25 (d, J ) 9.04 Hz, 1H), 8.07 (d, J )
8.8 Hz, 1H), 4.96 (q, J ) 7.08 Hz, 2H), 3.61 (s, 3H), 3.11 (s,
3H), 2.38-2.47 (m, 4H), 1.52-1.65 (m, 7H); 13C NMR (DMSO,
50 MHz) δ 173.2, 172.1, 157.9, 144.8, 139.1, 134.3, 129.2, 127.7,
125.7, 119.8, 116.0, 51.2, 47.2, 36.0, 24.1, 24.0, 22.0, 13.5.
2{[4-(Diethylamino)phenyl][4-(diethyliminio)cyclohexa-
2,5-dien-1-ylidene]methyl}-5-({[6-oxo-6-({2-[2-(2-{4-[2-oxo-
2-(6-oxo-5,6-dihydro-11H-pyrido[2,3-b]1,4benzodiazepin-
11-yl) ethylpiperazin-1-yl}ethoxy)ethoxy]ethyl}amino)-
hexyl]amino}sulfonyl)]-benzenesulfonate (4). A solution
of DIPEA (0.5 mL of 14.5 mM, 7.2 µmol) was added to a
suspension of the pirenzepine derivative 3 (2.2 mg, 2.67 µmol)
in CH3CN (1 mL). Solubilization was achieved with DMF (0.5
mL). A solution of succinimidyl ester 8 (2 mg, 2.65 µmol) in
2-[(E)-2-Anilinovinyl]-1-ethylquinolinium Iodide (17).
Quinaldine ethiodide 15 (500 mg, 1.67 mmol) and N,N′-
diphenylformamidine 16 (328 mg, 1.67 mmol) were vigorously

7858 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Tahtaoui et al.
CH3CN (1 mL) was added upon stirring at room temperature,
and the reaction was monitored by TLC for the appearance of
a polar product (MeOH/CH2Cl2 2/8, Rf 0.15). After 3 h at room
temperature, the reaction was stopped, and the solvents were
removed under reduced pressure. Semipreparative HPLC was
performed using a C18 Dionex Vydac column (22 mm × 250
mm; 300 Å; 15 µm) equilibrated in solvent A (H2O 70%/MeOH
30%/TFA 0.05%; v/v) and eluted with a linear gradient from 0
to 100% of solvent B (MeOH 100%/TFA 0.05%; v/v) in 38 min
at a flow rate of 10 mL/min. This allowed the determination
of a tr of 25.8 min for 4 and its isolation as a blue product (1.0
mg, 34% yield). Its purity (around 93%) was then confirmed
by two different analytical HPLC runs with absorbance
monitored at 220 and 600 nm. First, using a Zorbax Eclipse
C18 column (150 mm × 4.6 mm) equilibrated in solvent A
(MeOH 30%/H2O 70%/AcOH 0.5%; v/v), elution proceeded with
a linear gradient from 0 to 100% of solvent B (MeOH) in 40
min at a flow rate of 0.5 mL/min (tr ) 23.6 min). The second
system involved a Waters Nova-Pack C18 column (150 mm ×
3.9 mm; 60 Å; 4 µm) equilibrated in solvent A (H2O 90%/CH3-
CN 10%/TFA 0.05%; v/v) and eluted with a linear gradient
from 0 to 100% solvent B (H2O 10%/CH3CN 90%/TFA 0.05%;
v/v) in 60 min at a flow rate of 1 mL/min. A tr of 30.0 min was
found for 4. ESI-TOF m/z calculated 1108.50 (C57H74N9O10S2),
found 1108.44 (M + H)+, 554.70 [(M + 2H)+]/2.
Variations of this signal upon dye-labeled ligand addition
(usually 4 µL of a 250-fold concentrated DMSO stock) were
followed in real time. Occupancy curves were generated by
plotting amplitudes for fluorescence extinction (in percent) at
equilibrium as a function of ligand concentration. Kd values
and maximal FRET amplitudes were from data fitting to the
empirical Hill equation derived for saturation. Dissociation
traces were fit according to a monoexponential decay of
fluorescence to derive off-rate constants. Mathematical analy-
ses were done using Kaleidagraph 3.08 (Synergy Software).
Estimation for Donor-Acceptor Distances. The dis-
tance R0 (Fo¨rster radius) for 50% energy transfer efficiency
E, the spectral overlap integral J for the combined emission
of EGFP and dye absorbance, and the donor-acceptor separa-
tion R were calculated as previously described.9,13-14 Briefly,
R0 (Å) was calculated from the equation8 R0 ) 9790(κ2N-4ΦDJ)1/6
with κ2, N and ΦD parameters taken to be, respectively, 2/3,
1.4, and 0.66.
The distance R (Å) between EGFP and the dye moiety of
bound ligands was estimated using Fo¨rster’s equation:7 R )
R0(1/E - 1)1/6. The efficiency of fluorescence energy transfer
(E) was defined as the fractional decrease in specific EGFP
fluorescence due to ligand binding, that is, maximal FRET
amplitude (%) achieved at a saturating ligand concentration
corrected for nontransfected cell autofluorescence.
1-Ethyl-2-[(1E,3E)-3-(1-ethylquinolin-2 (1H)-ylidene)
prop-1-enyl]-6{[6-oxo-6-({2-[2-(2-{4-[2-oxo-2-(6-oxo-5,6-di-
hydro-11H-pyrido [2,3-b][1,4]benzodiazepin-11-yl)ethyl]-
piperazin-1-yl}ethoxy)ethoxy]ethyl}amino)hexanoyl]-
amino}] Quinolinium Iodide (5). To a solution of compound
19 (5 mg, 8.02 µmol) in anhydrous DMF (3 mL), BOP (4.61
mg, 10.4 µmol) and NMM (1.14 µL, 10.4 µmol) were added.
The mixture was stirred for 30 min at room temperature before
the addition of the derivative 3 (4.45 mg, 8.82 µmol). Stirring
was pursued for 12 h at room temperature. After AcOEt
addition, the precipitate was filtered off and washed with
AcOEt. HPLC purification of the colored probe 5 was per-
formed on a Zorbax reversed-phase C18 column (250 mm × 4.6
mm) using an isocratic elution mode (CH3CN 35%, H2O 65%,
TFA 0.1%; v/v) for 30 min at a flow rate of 1 mL/min (tr )
22.0 min). The second system involved a Waters Nova-Pack
C18 column (150 mm × 3.9 mm; 60 Å; 4 µm) and elution
conditions identical to those previously detailed for 4 and led
here to the determination of a tr of 38.3 min for compound 5.
MALDI-TOF m/z calculated 1073.40 (C55H64IN9O6), found
946.50 (M - I-)+.
Acknowledgment. This work was supported by the
Centre National de la Recherche Scientifique, the In-
stitut National de la Sante´ et de la Recherche Me´dicale,
the Universite´ Louis Pasteur, the Ministe`re de la
Recherche (ACI 2002), IFR 85, and Hoechst-Marion-
Roussel (fellowship to C.T.). We are grateful to Dr. J.
Garwood for critical reading of the manuscript and to
Dr. D. Bonnet and C. Antheaume for their valuable
contribution to NMR studies.
Supporting Information Available: A table listing the
formulas, molecular and isotopic weights, MALDI-TOF analy-
ses, and HPLC elution times for compounds 4, 5, 7, and 19
1
and H NMR and 2D-NOESY NMR spectra and analyses for
compound 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
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