Original design of fluorescent ligands by fusing BODIPY and melatonin neurohormone

Article abstract of DOI:10.1021/ml4004155

An original design and synthesis of fluorescent ligands for melatonin receptor studies is presented and consists in the fusion of the endogenous ligand with the fluorescent BODIPY core. Probes I-IV show high affinities for MT₁ and MT₂ melatonin receptors and exhibit fluorescence properties compatible with cell observation.

Full text of DOI:10.1021/ml4004155

Letter
pubs.acs.org/acsmedchemlett
Original Design of Fluorescent Ligands by Fusing BODIPY and
Melatonin Neurohormone
Jeremy Thireau,† Justine Marteaux,† Philippe Delagrange,‡ Francois Lefoulon,§ Laurence Dufourny,∥
́
́
Gerald Guillaumet,† and Franck Suzenet*,†
́
†Universite
‡Unite
de Recherches et Dec
§Technologie SERVIER, 27 rue Eugen
́
d’Orlea
́
ns, CNRS, ICOA, UMR 7311, F-45067 Orleans, France
ouvertes en Neurosciences, Institut de Recherches Servier, 78290 Croissy-sur-Seine, France
́
́
́
́
e Vignat, 45000 Orleans, France
∥UMR INRA-CNRS 7247-Univ. Tours-IFCE, PRC, Centre INRA de Tours, 37380 Nouzilly, France
S
* Supporting Information
ABSTRACT: An original design and synthesis of fluorescent
ligands for melatonin receptor studies is presented and consists
in the fusion of the endogenous ligand with the fluorescent
BODIPY core. Probes I−IV show high affinities for MT1 and
MT2 melatonin receptors and exhibit fluorescence properties
compatible with cell observation.
KEYWORDS: GPCRs, melatonin, fluorescence, molecular probes, BODIPY
activation. To offer alternative probes, we have developed a
luorescence is one of the most sensitive spectroscopic
Fmethods and many fluorescent ligands have been reported concept aiming at using the aromatic core of an endogenous
ligand as the source of fluorescence after slight chemical
modification and without loss of biological activity. Herein, we
report the design and synthesis of fluorescent ligands for
melatonin receptor studies thanks to the fusion of the
endogenous ligand with the fluorescent BODIPY core.
Melatonin presents in its chemical structure an indole ring
possessing fluorescent properties that are unfortunately
inappropriate for biological analysis due to interferences from
other biochromophores (such as tryptophan).10,11 Our
expertise on the melatonin structure/activity relationship12−16
prompts us to investigate the extension of the π-conjugation of
the indole scaffold at position C-2 in order to obtain
biologically compatible photophysical properties. The original
idea consists in the fusion of the pyrrole ring of melatonin with
one of the pyrrole rings of the highly stable and bright difluoro-
boraindacene (BODIPY) fluorophore17−19 (Figure 1). The
fluorescence of the resulting indole-based BODIPY was
expected as recently described by Zhao, Zhu, and co-
workers.20,21
for locating G-protein-coupled receptors (GPCRs), for study-
ing ligand/receptor interactions, and more generally for better
understanding their pharmacology and physiological proc-
ess.1−4 The history of fluorescent ligands is linked to the
development of commercially available fluorophores. Organic
dyes have been designed and synthesized to exhibit excitation
and emission wavelengths, which are compatible with biological
observation and are associated to ligands in conjugation
reactions. The addition of such a distinct fluorescent molecule
may alter both the chemical (while remaining within a
spectrum of lipophilicity to hydrophilicity) and pharmaco-
logical (affinity, functionality, etc.) properties of the resulting
fluorescent ligand that will modify its cellular behavior.
The melatonin receptors MT1 and MT2 are members of the
GPCR family. They are involved in the regulation of the
circadian rhythm and seasonal functions in mammals. They are
also implicated in many biological processes ranging from anti-
inflammatory to antioxidant effects including anti-Parkinson
effects5,6 and were recently reported as part of the mechanism
of action of the antidepressant agomelatine, an MT1 and MT2
receptor agonist and 5-HT2C antagonist.7,8 Despite the
discovery of the high affinity agonist and nonselective 2-
To attempt the fused melatonin-BODIPY core, 2-iodomela-
tonin 1,22−24 was converted into 2-formylmelatonin 2 by a
palladium catalyzed carbonylative coupling reaction in the
presence of tributyltin hydride in good yields using the
Fukuyama procedure.25 Condensation of the pyrroles with 2
[
125I]-MLT radioligand9 research on the pharmacology and the
functionality/physiological impact of melatonin receptors
suffers from the lack of selective probes for these receptors
due to their very low level of expression. Moreover, the main
disadvantages of this method are the radioactive hazards and
the limitations of studying the molecular dynamics of receptor
Received: October 15, 2013
Accepted: November 20, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Scheme 2. Synthetic Pathway to Molecules III and IV
Figure 1. Fused melatonin-BODIPY.
under traditional experimental conditions using acidic catalyst
(POCl3, HBr) was unable to furnish the desired product.
Considering the final difluoroborane complex, we envisaged the
direct activation of the carbonyl function in compound 2 with
boron trifluoroborate etherate.26 The Lewis acid was added
slowly at low temperature to the solution of 2-formylmelatonin
2, and after 15 min the pyrrole was added. Synthesis of the final
boron complex was achieved by adding triethylamine with six
extra equivalents of BF3·Et2O. The first two desired structures I
and II were isolated in 21% and 24% yield, respectively
(Scheme 1).
Scheme 1. Synthetic Pathway to Molecules I and II
Figure 2. Normalized absorption of compounds I (green), II (blue),
III (red), and IV (purple) in DMSO.
Table 1. Absorption Wavelengths and Molar Extinction
Coefficients of Compounds I−IV in DMSO
compd
absmax (nm)
ελmax (L·mol−1·cm−1
I
II
III
569
29544
IV
604
31230
515
512
)
31094
32970
Accentuation of the conjugation in BODIPY is known to
induce a red-shift in the excitation and emission wavelengths,
which is more compatible for biological observations.17−19,27,28
Such a π extension can be performed by introducing an aryl or
a hetaryl group at the 3 and/or 5 positions of the pyrrole
moiety using metal catalyzed reactions,29−32 the Knoevenagel
reaction,33,34 vicarious nucleophilic substitution (VNS),35 or by
modifying the substituents linked to the boron atom.36 For our
purpose, aryl and styryl groups were envisaged and pyrroles 3
and 4 were first synthesized. Condensation with 2-formylme-
latonin according to the previous protocol finally afforded the
desired boron complexes III and IV with extended π-
delocalization in 35 and 37% yield, respectively (Scheme 2).
The photophysical properties of these new dyes were
measured in dimethylsulfoxide. Absorption (Figure 2) and
molar extinction coefficients of compounds I−IV are reported
in Table 1.
Figure 3. Normalized fluorescence emission spectra properties of
compounds I (green), II (blue), III (red), and IV (purple) in DMSO
(for λexc, see Table 2).
nm. Compounds III and IV, with extra π-conjugation, show
excitation and emission bands at lower energy as expected.
These new indole-based BODIPYs present Stokes shifts
between 21 and 111 nm comparable with standard BODIPY
dye values.17−19
The binding affinities of the four fluorescent derivatives I−IV
were evaluated (Table 3) on human MT1 and MT2 receptors.
They all show good affinities: ligands I and IV present affinities
in the range of tens of nanomolar concentrations for the two
All four boron complexes show fluorescent properties. The
emission spectra (Figure 3) for I and II are around 490−540
B
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426
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67
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111
573
616
43
583
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21
λem (nm)
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MT1 Ki SEM (nM)
32
MT2 Ki SEM (nM)
I
5
10 0.7
96 20
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Leppaluoto, J. Iodinated Melatonin: Preparation and Characterization
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receptors, while ligand II is more selective for the MT2
receptor, and ligand III displays a larger MT1 receptor
selectivity.
In conclusion, by fusing the endogenous ligand of melatonin
receptors with the well-known and efficient fluorescent
BODIPY core, we have been able to design and isolate four
new condensed fluorescent probes with good melatonin
receptor affinities. Extension of the π-conjugation of ligands I
and II by coupling with an aryl (ligand III) or a styryl group
(ligand IV) induces a bathochromic shift with slight impact on
the affinity. Cellular imaging studies are currently under way.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the synthesis and the characterization
of ligand I−IV and intermediates, spectroscopic data, and
pharmacology. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(F.S.) Tel: + 33 (0) 2 38 49 45 80. Fax: + 33 (0)2 38 41 72
81. E-mail: franck.suzenet@univ-orleans.fr.
■
Funding
This work was supported by La Reg
LOIREMEL). During this study, J.T. and J.M. were supported
(Ph.D grants) by the Conseil General du Loiret (J.T.) and La
Region Centre (J.M., APR2012-LIFERMEL)
Notes
́
ion Centre (APR2009-
́
́
́
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank Dr. S. Poupart for her advice.
■
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Synthesis and Spectroscopic Studies. Org. Biomol. Chem. 2012, 10,
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