Please do not adjust margins
ChemComm
Page 2 of 5
DOI: 10.1039/C8CC00483H
COMMUNICATION
Journal Name
fluorescent probes. Recently, we reported a coumarin-based Table 1 ERα and ERβ relative binding affinity (RBAs)a
Compound
WAY-202196 1.62 ± 0.32 138.53 ± 6.64 85.51
FPNM
0.214 ± 0.06 17.31 ± 4.4 80.9
native fluorescence probe, which had high affinity for ER
without conjugating a fluorescent dye, however, expressed
low ERβ selectivity.23 In addition, as we know, ERα and ERβ
share high degrees of homology in their binding sites, their
binding pockets are nearly the same, with only L384 and M421
in ERα replaced by M336 and I373 in ERβ, respectively.24
Despite of the recent progress made on developing ERβ-
targeted fluorescent probes, the poor selectivity makes it
difficult to find fluorescent probes for ERβ imaging. Therefore,
how to harness the difference of ERα and ERβ is the key point
for the design of ERβ-selective fluorescent probes.
RBA ERα
RBA ERβ
β/α
aRelative binding affinity (RBAs) values are determined by
competitive radiometric binding assays and are expressed as
IC50estradiol/IC50
compound
× 100 ± the range or standard deviation
(RBA, estradiol = 100%).28
with pyridinium hydrochloride could give
7 and the final
compound was prepared by condensation of the aldehyde
with malononitrile in ethanol. The reaction could be
completed within 24 h with high yield (86%). (See ESI† for
synthesis details)
WAY-202196 (Fig. 1) is a well-studied ERβ selective and
latent fluorophore ligand,25 however, its short emission
wavelength and low fluorescence quantum limited its wider
use. Based on this compound, a long-wavelength fluorescent
probe FPNM with excellent fluorescence properties was
designed for ERβ imaging in live cells, which followed the
principle of the intermolecular charge transfer (ICT).26 Herein,
we introduced the dicyanomethylene substituent as the
electron acceptor, the phenol hydroxyl groups of lead
compound were selected as the electron donor. And the
donor-acceptor architectures were bridged by a conjugated π-
electron chain. Unlike the reported fluorophore-labeled
conjugate probes, the hydroxyl group of phenol and
dicyanomethylene group used in the design of the ERβ
selective fluorescent probe were also pharmacophores, and
always acted as important substituents for ER binding.27
Moreover, only one “π-electron bridge” was incorporated into
the lead compound to significantly reduce the molecular
weight, and with conjugation of a double bond, the HOMO-
LUMO energy gap of probe becoming smaller, resulting in a
significant red shift both in absorption and emission spectra.
We first tested the binding affinities of FPNM for both ERα
and ER by a competitive radiometric receptor-binding assay
β
and the results were reported in Table 1. These affinities were
presented as relative binding affinity (RBA) values, where E2
has an affinity of 100. For reference, the KD of estradiol is 0.2
nM for ERα and 0.5 nM for ERβ. As expected, FPNM showed
low binding affinity for ERα, but high affinity for ERβ. The RBA
values of FPNM were 0.21 and 17.31 for ERα and ERβ,
respectively, and the ERβ/ERα selectivity was as high as 80.9.
Compared to the parent compound WAY-202196 (RBA values
were 1.62 for ERα and 138.53 for ERβ; β/α was 85.51), FPNM
still retained high binding affinity and good selectivity for ERβ
(Table 1).
To gain insight into the binding nature of FPNM to ERβ,
molecular docking simulations were performed. Molecular
modeling showed that the phenol of FPNM mimicked the
estradiol A-ring,29 where it interacted with ERβ residues
Glu305, Leu 339, Arg 346 and a highly ordered water molecule.
In addition, the naphthol hydroxyl projects between helices 3
and 11, and dicyanomethylene group was directed towards
helice 8. The core scaffold, consisted with the phenyl and the
naphthalene ring, filled the remainder of the primarily
hydrophobic pocket, and FPNM would behave in a similar
affinity and selectivity for ERβ to the parent compound.
Naphthalene is the core of several highly fluorescent entities
Here we report the first fluorescence imaging probe, FPNM
,
which shows preferable binding affinity to ERβ over ERα and is
suitable for ERβ imaging in living cells.
The synthesis of FPNM is presented in Scheme 1. To
construct the target molecule, tetralone
1
was converted to
, which was aromatized with Pd/C in p-
, dibromination of latter followed by
the cyano derivative
cymene to afford
2
3
including our target compound FPNM 30, 31
the absorption and
,
selective debromination leading to the key intermediate
Subsequent elaboration of the cyano group to the aldehyde
derivative , which coupled under Suzuki conditions led to the
phenylnaphthalenes . Finally, treatment of the intermediate
4.
fluorescence properties of FPNM were evaluated. As
anticipated, with the electron-deficient acceptor as the end
5
6
Scheme 1 Synthesis of probe FPNM. Reagents and conditions:
(a) TMSCN, ZnI2; (b) 10% Pd/C, p-cymene, reflux; (c) (i) Fig. 2 Model of FPNM bound to ERβ. Computer-developed
Br2/AcOH, (ii) SnCl2; (d) DIBAL; (e) ArB(OH)2, Pd(PPh3)4, K2CO3, model of FPNM bound to ERβ with the conserved H-bond to
toluene, 120 °C , 24 h; (f) Pyridine hydrochloride, 190 °C, 2 h; Glu 305, Leu 339, Arg 346 and an ordered water molecule, and
(g) Pyridine hydrochloride, EtOH, 60 °C, 24 h.
the core scaffold fills the remainder of the primarily
hydrophobic pocket.
Chem. Commun.
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins
Hu, Zhiye
