High affinity fluorescent ligands for the estrogen receptor

Article abstract of DOI:10.1002/ejoc.201403489

Fluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the deve

Full text of DOI:10.1002/ejoc.201403489

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DOI: 10.1002/ejoc.201403489
High Affinity Fluorescent Ligands for the Estrogen Receptor
Frank Abendroth,[a] Marthe Solleder,[b] Dorothea Mangoldt,[c] Pia Welker,[c] Kai Licha,[c]
Marcus Weber,[b] and Oliver Seitz*[a]
Keywords: Click chemistry / Hormones / Fluorescence / Fluorescent probes / Receptor–ligand interactions / Molecular
simulation / Cyanines
Fluorescent binders of the estrogen receptor (ER) are used in
binding assays and in detection or imaging studies. However,
fluorescence labelling of ER ligands usually leads to substan-
tial decreases in binding affinity. In this study, we describe
the development of high affinity fluorescent ER ligands.
Cyanine dyes of the MiDye series were directly attached to
the SERMs 4-hydroxytamoxifen (OHT) and raloxifene (Ral);
linkers were deliberately omitted. This approach yielded
conjugates with ERα binding affinities superior to the natural
ligand estradiol. The OHT- and Ral-MiDye conjugates
emitted in the 600–800 nm range. First round staining experi-
ments showed that the conjugates, but not the dyes alone,
accumulate in cells expressing estrogen-binding receptors.
Introduction
Estrogen receptors (ER) control proliferation and differ-
entiation of target tissues, and ER overexpression is often
associated with carcinomas of the breast, endometrium,
and ovary.[1] Compounds such as tamoxifen, 4-hydroxy-
tamoxifen or raloxifene (Figure 1, a) modulate ER-medi-
ated signaling enabling therapeutic intervention in a diverse
set of clinical indications. The importance of ER-mediated
signalling to human health has fueled the development of
fluorescent ER ligands, which are useful for binding assays
and ER detection/imaging. Many previously published ER
imaging probes contain the 17-α-[4-aminomethyl-phenyl-
ethynyl]-estra-1,3,5(10)-triene-3–17-β-diol core structure
(E2), providing useful ER affinity when bulky dyes were
appended via the distal end (Figure 1, b).[2] In addition,
fluorescent dyes have been tethered to the ER ligands 4-
hydroxytamoxifen (OHT-L-R in Figure 1, b)[3] and cyclo-
fenil.[4] In these and all other cases, the affinities for the
targeted ER are lower than the binding affinity of the natu-
ral ligand.[5]
The ER binds estrogen-like ligands as a dimer. We re-
cently reported the DNA-based spatial screening of the ER
and observed a bimodal distance–affinity relationship.[6]
High affinity was obtained when the ER ligands were pre-
sented on the DNA scaffold with distances that matched
[a] Department of Chemistry, Humboldt University Berlin,
Brook-Taylor-Straße 2, 12489 Berlin, Germany
E-mail: oliver.seitz@chemie.hu-berlin.de
http://www.chemie.hu-berlin.de/forschung/seitz
[b] Zuse Institute Berlin,
Takustraße 7, 14195 Berlin, Germany
[c] Mivenion GmbH,
Robert-Koch-Platz 4, 10115 Berlin, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201403489.
Figure 1. a) Estrogen receptor ligands and fluorescent conjugates
used b) previously and c) in this study.
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the distance between the two consensus estrogen binding 7:3 Z/E mixture. The electron rich alkene rapidly isomerized
sites observed in crystal structures. In addition, un- to a 1:1 mixture of Z/E isomers and upon nucleophilic dis-
expectedly high binding affinities were measured for ligand– placement with sodium azide, tamoxifen-azide OHT-N3
ligand distances shorter than 23 Å, too short to bridge the was formed.
two canonical estrogen binding sites in the ER dimer. This
behaviour was attributed to the presence of a second bind-
ing site within each ER monomer. In subsequent distance–
affinity studies, ethylene glycol-based spacers were used.[7]
Again, the highest binding affinities were observed for those
agents containing very short tethers. This was described as
being attributable to an intramolecular binding mode. Of
note, the interaction of ER ligands with “non-canonical”
binding sites is known from crystal structure analyses.[8]
Given the possibility for “non-canonical” interactions
between hydrophobic ER ligands and an additional binding
site, we inferred that hydrophobic tagging of ER ligands
may provide opportunities for the design of high affinity
fluorescent ER ligands. We postulated that hydrophobic in-
teractions between the tag and the additional binding patch
might compensate for losses in ER binding affinity usually
observed following conjugation of ER ligands. Here we re-
port the synthesis and fluorescence microscopic studies of
fluorescently labelled ER ligands based on 4-hydroxy-
tamoxifen and raloxifene. We show that the hydrophobicity
of the appended tag positively influences ER binding. This
provides, for the first time, fluorescent ER ligands that have
higher binding affinities than the endogenous ligand
estradiol.
Results
The hydrophobic nature of interactions at the coactivator
binding site called for hydrophobic fluorescent tags whereas
the rather small distance between the two sites (Ͻ 23 Å)
suggested tethering via short linkers. Yet, given the plas-
ticity of the ER ligand binding domain, we considered that
binding enhancements can only be obtained when the
and c) hydrophobic tagging.
putative auxiliary binding patch remains accessible follow-
Scheme 1. Synthesis of a) raloxifene azide and b) tamoxifen azide
ing ER ligand binding to the canonical binding pocket.
Azidation of putative ER ligands Ral-N3 and OHT-N3
Consequently, we tethered hydrophobic groups to known enabled facile attachment of tags based on 9-substituted
ER ligands 4-hydroxytamoxifen and raloxifene (Figure 1, acridine (Acr), anthracene (Ant) and 1-substituted pyrene
c). Both compounds belong to a class of so-called selective (Pyr) derivatives. For this purpose, the tag azides Tag-N3
estrogen receptor modulators (SERMs) which are currently (prepared from chloromethylated precursors by chloride–
used to treat ER-related diseases. Notably, the ER confor- azide exchange) were exposed to a large excess of bisprop-
mation induced by the two SERMS differs from the confor- argyl ether in the presence of copper(I) bromide (Scheme 1,
mation induced by estradiol.[9]
c). This provided the desired mono-functionalized dyes and
The aliphatic alcohol in raloxifene building block Ral- small amounts of the symmetric bis-triazoles (Figure S1).
OH was converted to an alkyl chloride upon treatment with Copper-mediated dipolar cycloaddition reactions with the
triphenylphoshine and tetrachloromethane (Scheme 1, a); SERM-azides furnished SERM-dye conjugates SERM-L-
no attempt was made to optimize this Appel reaction. Tag in 50–80% overall yield after column chromatography.
Rather, the alkyl chloride was forwarded to the next reac-
In vitro binding studies with the estrogen receptor alpha
tion which involved the nucleophilic displacement of chlor- (ERα) revealed substantial losses in binding affinity when
ide with sodium azide to yield raloxifen-azide Ral-N3 in the tags were added to 4-hydroxytamoxifen (Table 1).
quantitative yield. Rapid access to the azido-modified Nevertheless, the loss of binding affinity was lower for
tamoxifen building block was provided by a McMurry cou- pyrene containing compound OHT-L-Pyr than for conju-
pling between a chloroethylated benzophenone derivative gates containing anthracene (OHT-L-Ant) or acridine
and excess propiophenone (Scheme 1, b). The reaction (OHT-L-Acr). Careful assessments of the raloxifene conju-
yielded the corresponding triarylbutene intermediate in a gates and their ER binding characteristics showed that, as
2
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High Affinity Fluorescent Estrogen Receptor Ligands
anticipated, binding affinity increased with increasing
The SERM-MiDye conjugates were more soluble in
hydrophobicity (see clogP values in Table 1). Notably, the water than the corresponding triazole-linked SERM conju-
raloxifene-pyrene conjugate (Ral-L-Pyr) displayed a relative gates. Binding experiments with raloxifene containing
binding affinity similar to that of parent compound Ral.
probes revealed remarkable improvements in the relative
binding affinities (RBAs) (Table 2, Figure S3). Of note, the
RBAs of the Ral-MiDye conjugates were higher than for
free raloxifene. Conjugation with the heptamethine dyes
DMITCC and ITCC as well as with the cationic penta-
methine dye DMIDCC afforded compounds with ER bind-
ing affinities superior to that of estradiol. To the best of
our knowledge, such high binding affinity of fluorescent ER
ligands is without precedence in the literature. By compari-
son, the OHT-MiDye conjugates showed less spectacular
binding affinities; the binding affinities for these agents
were lower than for unconjugated 4-hydroxytamoxifen.
However, it is interesting to note that both raloxifene and
the 4-hydroxytamoxifen cores experienced up to a six-fold
increase in RBA when the bisimidazole ether-linked
Table 1. Relative binding affinities (RBA)[a] and clogP values[b] of
SERM conjugates.
RBA
[%]
RBA
[%]
clogP
clogP
OHT
90[c]
6
13
13
6.15
6.98
8.06
8.52
Ral
34
17
25
36
6.86
6.45
7.73
7.99
OHT-L-Acr
OHT-L-Ant
OHT-L-Pyr
Ral-L-Acr
Ral-L-Ant
Ral-L-Pyr
[a] RBA for ERα relative to estradiol. [b] Values calculated using
Chemdraw Ultra. [c] Based on 50:50 E,Z-mixtures.
The initial results confirmed the positive influence of
hydrophobic tags on ER ligands such as 4-hydroxytamox-
ifen or raloxifene and their binding to the ER. To tether
fluorescent labels enabling imaging studies in complex
matrices we sought a strategy that would avoid reaction
steps after introduction of the costly labels. Therefore,
conventional coupling of dye-bourne carboxylic acids with
raloxifene or tamoxifen amino groups was envisioned and
assigned as the preferred approach. We used monofunc-
tional cyanine dyes of the “MiDye” series which offer diver-
sity in their hydrophobic character and emission wave-
lengths spanning the red to near-infrared region (650–
800 nm). SERM-azides Ral-N3 and OHT-N3 were reduced
to the SERM-amines and subsequent coupling with the
MiDye-carboxylic acids was performed using TSTU as the
activating reagent in the presence of diisopropylethylamine
(Scheme 2). After HPLC purification SERM-MiDye conju-
gates of interest were obtained in 55–71% yield.
acridine in Ral-L-Acr and Tam-L-Acr was replaced by ethyl
amido-linked cyanines in Ral-ITCC and Tam-DMITCC.
Table 2. Relative binding affinities[a] of SERM-MiDye conjugates
and E2-FITC.
RBA [%][b]
RBA [%]
OHT-DMIDCC
33Ϯ4
Ral-DMIDCC
Ral-IDCC
105Ϯ15
68Ϯ12
OHT-DMITCC
OHT-ITCC
E2-FITC
35Ϯ3
21Ϯ2
14Ϯ7
Ral-DMITCC
Ral-ITCC
122Ϯ23
127Ϯ12
[a] RBA for ERα relative to estradiol. [b] Based on 50:50 E,Z-mix-
tures.
A molecular dynamics analysis was carried out to ex-
plore interactions that could induce affinity enhancements
to ER ligands such as raloxifene. In this theoretical study
we compared the non-conjugated SERM raloxifene (Ral)
with its tagged MiDye derivative Ral-ITCC. In accordance
with Durmaz et al. we estimated free energy differences for
the binding.[10] This analysis revealed that binding of Ral-
ITCC to the ER released 60 kJ/mol more free energy than
the binding event involving non-conjugated Ral. This is
consistent and supportive of the experimental results. Fig-
ure 2 depicts graphical representations of ligand–ER com-
plexes displaying the strongest interactions. The molecular
dynamics analysis revealed the expected plasticity of the ER
structure. The conformation of the ER in complex with
Ral-ITCC differs from that of the complex formed with Ral;
this is most apparent in ER regions close to the binding
site. Of note, ER helix 12 (highlighted in light blue in Fig-
ure 2) seems to take part in additional interactions with the
hydrophobic tag (heptamethine dye of Ral-ITCC high-
lighted in yellow). Not surprisingly, this may explain why
Ral-ITCC binds to ERα more tightly than Ral.
We next evaluated the fluorescence properties of the
SERM-MiDye conjugates. Regardless of the SERM and
overall charge, the pentamethine dyes IDCC and DMIDCC
emitted at λmax = 670 nm and furnished quantum yields in
Scheme 2. Conjugation of tamoxifen and raloxifene with cyanine
dyes of the MiDye series. TSTU, O-(N-succinimidyl)-N,N,NЈ,NЈ-
tetramethyluronium tetrafluoroborate.
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Figure 2. Docking of the ligands (orange) a) Ral and b) Ral-ITCC
to the ligand binding domain of ERα (gray). The hydrophobic tag
in Ral-ITCC (yellow) picks up interactions with the C-terminal
helix 12 (light blue).
a range of φ = 0.25–0.29 provided that BSA was added
(Table S2). The heptamethine dyes ITCC and DMITCC
also showed the expected quantum yield φ = 0.09 and
emission maximum λmax = 770 nm at both tamoxifen and
raloxifene cores. The rather low fluorescence quantum
yields in the absence of BSA as well as the concentration
dependency of the fluorescence/absorbance spectra (Figure
S2) are indicative of hydrophobic interactions that induce
aggregation at µM concentrations. This behavior appeared
to be less problematic with sulfonated IDCC and ITCC
containing compounds. Consequently, these dyes were
Figure 3. Staining of MCF-7 cells with a) Ral-IDCC or b) IDCC.
Conditions: 30 min incubation with 100 nM IDCC-COOH or Ral-
IDCC, two washes with PBS buffer (please note a 15-fold higher
exposure time (8880 ms) was used for b) IDCC, see SI, S8 for de-
tails). Co-staining of c, cЈ MCF-7 and d, dЈ HEK cells with DAPI
(c), d)) and Ral-IDCC (cЈ, dЈ) Conditions: 30 min incubation with
5 nM Ral-IDCC (see SI for details). Images were acquired by using
a Zeiss AXIO observer microscope and a 40ϫ objective.
To assess the specificity of ER staining we performed
selected for staining experiments (vide infra). The high ER displacement experiments. Unpermeabilized MCF-7 cells
affinity of the SERM-MiDye conjugates suggested that were treated first with Ral-IDCC and then with excess
meaningful staining experiments could be performed at estradiol E2. Confocal fluorescence microscopy after cell
sub-micromolar concentrations when aggregation of the fixation revealed strong staining of cytosolic areas when the
fluorescent ER ligands is hampered.
cells were incubated exclusively with Ral-IDCC (Figure 4,
In fluorescence microscopy experiments with SERM- a). In addition, nuclear staining was observed under these
IDCC and -ITCC conjugates it was the aim to investigate conditions. After E2 treatment the intensity of IDCC emis-
staining of intracellular ERs. First we compared the uncon- sion was markedly reduced (compare Figure 4, a and aЈ).
jugated IDCC dye with the Ral-IDCC conjugate in the ER- The remaining IDCC signals showed a punctuate localiza-
positive MCF-7 cell line. Despite the low concentration tion pattern, which may indicate segregation. Fluorescence
(5 nM) Ral-IDCC conjugate provided high contrast in non- activated cell sorting showed that the Ral-MiDye ligand
permeabilized cells indicative of intracellular enrichment was able to displace E2-FITC indicative of receptor binding
(Figure 3, a). By contrast, the attempted staining of MCF-7 (Figure S4). We also explored staining of raloxifene binding
cells with the SERM-free dye failed (Figure 3, b). A further receptors in SKBR-3 cells. This ER-negative cell line ex-
control experiment involved a comparison between MCF-7 presses the non-nuclear, G-protein coupled estrogen recep-
cells and HEK cells. The latter do not express ERs and, tor GPER. Confocal fluorescence microscopy showed in-
indeed, staining with Ral-IDCC failed (compare Figure 3, tense IDCC emission when the SKBR-3 cells were incu-
cЈ and dЈ).
bated with Ral-IDCC (Figure 4, b). Competition with E2
4
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High Affinity Fluorescent Estrogen Receptor Ligands
resulted in a distinct reduction of IDCC intensity (Figure 4, be improved by attachment of a tag provides clues for
bЈ, for FACS analysis see Figure S5).
designing improved estrogen analogues.
Fluorescence microscope images revealed that the Ral-
IDCC conjugate enriches in MCF-7 cells (ER+, GPER+)
and SKBR-3 cells (ER–, GPER+) but not in HEK cells
(ER–, GPER–). In MCF-7 cells the confocal fluorescence
microscope images showed strong fluorescence in the cyto-
sol and, by comparison, weaker fluorescence in the nucleus.
This stands in contrast to the known preference for nuclear
localization by the ER. A similar trend has been previously
noted when non-permeabilized cells were treated with a
BODIPY-labeled 4-hydroxytamoxifen.[3] The authors at-
tributed the noted results to the rather modest potency and
potential aggregation of probes at the high concentrations
required to provide measurable fluorescence. Yet, our con-
jugates are characterized by high binding affinities and en-
able fluorescence measurements at low nanomolar concen-
trations. Still, nuclear staining remained comparatively
weak, raising questions about staining specificity. The
marked reduction in observable staining after incubation
with E2 suggests that enrichment of Ral-IDCC in MCF-7
(and SKBR-3) cells must have significant contributions
from specific receptor binding interactions. Although we
did not optimize for completeness of displacement, the
punctuate pattern of the remaining Ral-IDCC signals
Figure 4. Confocal fluorescence microscopy images after staining
of non-permeabilized a, aЈ MCF-7 cells and b, bЈ SKBR-3 cells suggests that care has to be taken when hydrophobic ER
with a), b) Ral-IDCC (red) followed by aЈ, bЈ incubation with ex-
ligands are used in localization studies. These data do not
cess E2 and subsequent fixation. Costaining was performed with
exclude the possibility that contributions from non-specific
DAPI (blue). Conditions: 10 min incubation with 100 nM Ral-
interactions play a role since E2 did not completely displace
IDCC, after 10 min cells were incubated with 10 μm E2 for 30 min
Ral-IDCC. Furthermore, non-specific staining is, in part, a
function of cell type as evidenced by the improved efficiency
of Ral-IDCC displacement in SKBR-3 cells. Such findings
clearly warrant further investigations of affinity-enhanced
raloxifene-dye conjugates and alternative staining protocols.
(aЈ, bЈ). Images were acquired by using a Zeiss LSM5 Exciter
microscope and a 40ϫ objective.
Discussion
Assuming similar cell permeability, it appears plausible
to correlate the extent of probe enrichment within target
cells with the probeЈs affinity for cognate receptors. In this
regard, the loss of ER affinity upon fluorescence labeling
of ER ligands is a nuisance. Our results suggest that
Conclusions
In this study, we developed high affinity fluorescent
hydrophobic tagging of ER ligands with short linkers can ligands for the estrogen receptor The design of the ligand
compensate, to an extent, for losses in ER binding conjugates was guided by reports of a secondary binding
affinity due to such fluorophore tagging. Notably, labeled site for hydrophobic groups in the vicinity of the consensus
compounds such as raloxifene-MiDye conjugate Ral-ITCC estrogen binding site. We developed a synthetic route that
are characterized by a four-fold higher binding affinity than enables rapid access to hydrophobically tagged SERMs. In
either the unlabeled compound or estradiol, the endo- vitro binding measurements showed that the affinity for
genous ER ligand. Our results also showed that hydro- ERα correlated well to hydrophobicity of the SERM conju-
phobic tagging was more successful for raloxifene than for gates. Notably, the conjugation of Cy-like MiDyes with
4-hydroxytamoxifen conjugates. It is known that the confor- raloxifene furnished conjugates that had higher ER affinity
mation of the ER adapts to specific ligands and it is con- than the natural ligand estradiol. ER–ligand binding affin-
ceivable that the conformation induced by raloxifene pro- ities of this magnitude are without precedence. Fluores-
vides patchy binding surfaces for the MiDyes that are not cence emission spectra of the cell permeable SERM-MiDye
generated by 4-hydroxytamoxifen–ER binding. Alterna- conjugates span the 600–800 nm range and may facilitate
tively, the Ral and 4OHT-dye conjugates may experience measurements in live cells and tissues. Preliminary fluores-
different extents of intramolecular hydrophobic interac- cence microscopy experiments revealed that MiDye-labeled
tions,[7b] which may alter ER affinity. Regardless of the raloxifene undergoes significant enrichment in unpermeabi-
mechanisms involved, the fact that a given ER ligand can lized cells expressing E2-binding receptors.
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chloride (1.83 mL, 16 mmol) within 20 min. The mixture was re-
fluxed for 2 h. To the dark-grey-purple suspension was added in
one portion a solution of propiophenone (1.62 g, 12 mmol). Sub-
sequently, 4-chloroethoxy-4Ј-hydroxybenzophenon (1.1 g, 4 mmol)
in dry THF (23 mL) was added in one portion. Refluxing was con-
tinued for 5 h. After cooling to room temperature the reaction was
quenched by the addition of a solution of 10% sodium carbonate
[w/v] in water. The mixture was stirred for 10 min and filtered
through celite. The filtrate was evaporated to dryness. The residue
was dissolved in EtOAc (200 mL) and washed with brine (2ϫ
100 mL). The organic layer was dried with sodium sulfate, the sol-
vent was removed in vacuo and the oily residue was purified by
column chromatography (E/hexane, 3:7). This yielded 1.15 g (76%)
of a highly viscous liquid. Rf = 0.50 (hexane/EtOAc, 7:3), HRMS
(ESI, negative): m/z = calcd. 377.1308 (for [C24H22ClO2]–), found
Experimental Section
Synthesis of SERM Azides. Ral-OH: To a stirred solution of ethyl-
ene glycol (2.4 g, 39 mmol, 7.2 equiv.) in 2 mL dry DMF under
argon was carefully added sodium hydride (658 mg, 26 mmol,
5.5 equiv.). After completion of the exothermic reaction, the mix-
ture was stirred at 90 °C for additional 20 min before heating was
discontinued. To the suspension was added a solution of 3-[p-
fluorobenzoyl]-6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene
(2 g, 5.5 mmol, 1 equiv.) dissolved in anhydrous DMF (5 mL). Af-
ter 18 h of stirring at 90 °C, the solution was allowed to reach room
temperature before distilled water (100 mL) was added. The pH
was adjusted to pH 3 through addition of diluted HCl. The mixture
was extracted with EtOAc (100 mL). The organic phase was sepa-
rated and washed with brine (2ϫ 50mL) and dried with sodium
sulfate. After evaporation of the solvent and chromatographic sepa-
ration (silica gel 60, CH2Cl2/MeOH [9:1]), 1.9 g (85%) of a yellow
foam was obtained. Rf = 0.38 (CH2Cl2/MeOH, 9:1), HRMS (ESI,
1
377.1314. H NMR (300 MHz, [D6]DMSO): δ = 9.42 (s, 0.23 H),
9.17 (s, 0.78 H), 7.18 (dd, J = 7.89, 7.1 Hz, 2 H), 7.10 (ddd, J =
8.49, 3.31, 2.2 Hz, 4.5 H), 6.99–6.93 (m, 2 H), 6.74 (dd, J = 14.4,
8.7 Hz, 1 H), 6.64–6.56 (m, 2 H), 6.40 (d, J = 8.7 Hz, 1.6 H), 4.29–
4.23 (m, 1.6 H), 4.11–4.08 (m, 0.47 H), 3.98–3.92 (m, 1.56 H), 3.88–
3.82 (m, 0.47 H), 2.40 (td, J = 14.80, 5.70, 5.7 Hz, 2 H), 0.84 (t, J
= 7.4 Hz, 3 H) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 156.56,
156.03, 155.71, 155.17, 142.10, 140.00, 139.75, 137.70, 136.13,
135.89, 133.76, 133.47, 131.39, 131.29, 130.12, 130.03, 129.29,
127.81, 127.76, 126.44, 125.90, 125.84, 125.76, 114.88, 114.21,
114.12 113.33, 67.76, 43.07, 28.39, 13.34 ppm.
positive): m/z
=
calcd. 407.0953 (for [C23H18O5S]+), found
1
407.0948. H NMR (300 MHz, CD3CN): δ = 7.69 (d, J = 8.99 Hz,
2 H), 7.42–7.27 (m, 4 H), 7.22 (d, J = 8.7 Hz, 2 H), 6.87 (dd, J =
8.8, 2.3 Hz, 1 H), 6.81 (d, J = 9.0 Hz, 2 H), 6.67 (d, J = 8.7 Hz, 2
H), 4.01 (dd, J = 7.85, 3.4 Hz, 2 H), 3.77 (dd, J = 9.41, 4.9 Hz, 2
H), 3.09 (d, J = 5.7 Hz, 1 H) ppm. 13C NMR (75 MHz, CD3CN):
δ = 193.87, 164.14, 158.29, 155.85, 142.67, 140.85134.14, 132.98,
131.27, 131.15, 125.97, 124.68, 116.36, 115.84, 115.12, 107.98,
70.72, 61.01 ppm.
OHT-N3: A solution of OHT-Cl (67 mg, 177 μmol) and sodium
azide (115 mg, 1.77 mmol) dissolved in DMF (500 μL) was stirred
for 20 h at 100 °C. After cooling to room temperature the solution
was diluted with water (50 mL) and EtOAc (50 mL). The organic
layer was collected, washed with brine (50 mL) and dried with so-
dium sulfate. The volatiles were removed and the residue was puri-
fied by column chromatography (EtOAc/hexane, 3:7) yielded 64 mg
(95%) of a white solid. Rf = 0.51 (hexane/EtOAc, 7:3), HRMS
(ESI, negative): m/z = calcd. 384.1712 (for [C24H22N3O2]–), found
Ral-Cl: To a solution of Ral-OH (1.63 g, 4.0 mmol) and tri-
phenylphosphine (1.15 g, 4.4 mmol) in dry THF (50 mL) under ar-
gon was added CCl4 (20 mL) over the course of 30 min. The solu-
tion was refluxed for 16 h under argon. The volatiles were removed
in vacuo. Column chromatography (CH2Cl2/MeOH, 9:1) of the res-
idue provided 680 mg (40% yield) of a yellow foam. Rf = 0.35
(CH2Cl2/MeOH, 9:1), HRMS (ESI, negative): m/z
= calcd.
423.0458 (for [C23H16ClO4S]–), found 423.0464. 1H NMR
(300 MHz, CD3CN): δ = 7.68 (d, J = 9.0 Hz, 2 H), 7.40 (d, J =
8.8 Hz, 1 H), 7.33 (d, J = 2.2 Hz, 1 H), 7.21 (d, J = 8.7 Hz, 2 H),
6.88 (dd, J = 8.76, 2.3 Hz), 6.79 (d, J = 9.0 Hz, 2 H), 6.67 (d, J =
8.7 Hz, 2 H), 4.25–4.12 (m, 2 H), 3.79–3.83 (m, 2 H) ppm. 13C
NMR (75 MHz, CD3CN): δ = 193.84, 163.31, 158.28, 155.84,
142.98, 140.85, 134.10, 133.01, 131.69, 131.18, 124.69, 116.33,
115.84, 115.12, 107.99, 69.17, 43.30 ppm.
1
384.1718. H NMR (300 MHz, CD3CN): δ = 7.14–7.02 (m, 1 H),
6.99 (d, J = 8.7 Hz, 1 H), 6.88 (d, J = 8.8 Hz, 1 H), 6.73 (dd, J =
8.8, 1.1 Hz, 2 H), 6.62 (d, J = 8.8 Hz, 1 H), 6.51 (d, J = 8.9 Hz, 1
H), 6.37 (d, J = 8.8 Hz, 1 H), 4.12 (t, J = 4.91, 4.91 Hz, 1 H), 3.95
(t, J = 4.91, 4.9 Hz, 1 H), 3.53 (t, J = 4.9 Hz, 1 H), 3.43 (t, J =
4.9 Hz, 1 H), 2.59–2.32 (m, 2 H), 0.87 (t, J = 7.5 Hz, 3 H) ppm.
13C NMR (75 MHz, [D6]DMSO): δ = 158.03, 157.20, 156.57,
155.74, 143.59, 143.57, 142.07, 141.93, 138.92, 137.60, 137.49,
136.10, 135.99, 132.59, 131.27, 130.57, 128.66, 128.64, 126.89,
115.77, 115.07, 115.00, 114.21, 67.92, 67.65, 50.92, 50.84, 29.64,
29.59, 13.71 ppm.
Ral-N3: To a solution of Ral-Cl (345 mg, 0.813 mmol) in DMF
(1 mL) was added sodium azide (NaN3) (260 mg, 4 mmol). The
mixture was heated to 100 °C for 20 h. After cooling to room tem-
perature the solution was diluted with water (50 mL) water and
EtOAc (50 mL). The organic layer was collected, washed with brine
(50 mL) and dried over sodium sulfate. The volatiles were removed
and dry residue was dissolved in a minimum amount of CH2Cl2/
MeOH, 9:1 and purified by flash column chromatography
(CH2Cl2/methanol, 9:1). This afforded 322 mg (92% yield) of a yel-
low solid. Rf = 0.36 (CH2Cl2/MeOH, 9:1), HRMS (ESI, negative):
m/z = calcd. 430.0862 (for [C23H16N3O4S]–), found 430.0868. 1H
NMR (300 MHz, CD3CN): δ = 7.71 (d, J = 8.96 Hz, 2 H), 7.41
(d, J = 8.77 Hz, 1 H), 7.34 (d, J = 2.20 Hz, 1 H), 7.26 (s, 1 H),
7.23 (d, J = 8.69 Hz, 2 H), 7.18 (s, 1 H), 6.88 (dd, J = 8.77, 2.31 Hz,
1 H), 6.83 (d, J = 8.94 Hz, 2 H), 6.68 (d, J = 8.71 Hz, 2 H), 4.16–
4.02 (m, 2 H), 3.58–3.52 (m, 2 H) ppm. 13C NMR (75 MHz,
CD3CN): δ = 193.79, 163.41, 158.32, 155.88, 142.95, 140.88,
134.16, 133.01, 131.75, 131.21, 126.00, 124.72, 116.36, 115.86,
115.16, 107.99, 68.17, 50.71 ppm.
General Procedure for Dual Click Labelling: To a solution of
Tag-azide (300 μmol) (9-azidomethylacridine or 9-azidomethylan-
thracene or 1-azidomethylpyrene) in THF (2 mL) were added bis-
propargyl ether (2.05 mL, 20 mmol), 2,6-lutidine (3.5 μL, 30 μmol)
and copper(I) bromide (1.43 mg, 10 μmol). After overnight stirring
under argon the solvent was removed in vacuo. The residue was
dissolved in CH2Cl2 (50 mL) and washed with 0.1 m hydrochloric
acid (2ϫ 50 mL) and brine (2ϫ 50 mL). The organic layer was
dried with sodium sulfate and concentrated to dryness. The residue
was purified by flash column chromatography (CHCl3/MeOH, 99:1
to 9:1).
The Tag-L (200–300 μmol) was dissolved in THF (900 μL) and
SERM-azide (100 μmol) (OHT-N3 or Ral-N3) was added. Sub-
sequently, a solution of 50 mm TBTA and 10 mm copper(I) bromide
in 1:1 DMSO/water (100 μmol) was added. The mixture was stirred
at ambient temperature for 16 h. After quenching with an aqueous
OHT-Cl: To a well stirred solution of zinc (2 g, 32 mmol) in dry
THF (34 mL) under argon was added dropwise titanium tetra-
6
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Date: 19-02-15 17:46:33
Pages: 11
High Affinity Fluorescent Estrogen Receptor Ligands
NaSH solution [50 μL, 10% aq., (w/v)] the black precipitate formed
was removed by filtration. The filtrate was concentrated to dryness
and the residue purified by column chromatography (CH2Cl2/
MeOH, 9:1).
(d, J = 8.9 Hz, 2 H), 7.34 (d, J = 2.2 Hz, 1 H), 7.23 (d, J = 8.8 Hz,
1 H), 7.16 (d, J = 8.60 Hz, 2 H), 6.89 (d, J = 9.0 Hz, 2 H), 6.84
(dd, J = 8.8, 2.3 Hz, 1 H), 6.71 (s, 2 H), 6.66 (d, J = 8.6 Hz, 2 H),
4.70 (t, J = 5.0 Hz, 2 H), 4.47 (s, 2 H), 4.45 (s, 2 H), 4.40 (t, J =
5.0 Hz, 2 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 192.43,
161.90, 157.71, 155.31, 148.24, 143.61, 143.51, 140.37, 139.11,
136.73, 132.12, 131.67, 130.18, 130.05, 129.76, 129.58, 129.51,
126.94, 124.66, 124.48, 124.37, 124.11, 123.63, 123.18, 115.60,
115.10, 114.45, 107.01, 66.22, 62.56, 62.38, 48.59, 44.48, 40.34 ppm.
OHT-Acr: 32 mg (E/Z-mixture, 1:1), 75%, Rf = 0.60 (CH2Cl2/
MeOH, 9:1). HRMS (ESI, negative): m/z = calcd. 712.3042 (for
[C44H38N7O3]–), found 712.3043. 1H NMR (500 MHz, [D6]-
DMSO): δ = 9.43 (s, 1 H), 9.18 (s, 1 H), 8.67 (d, J = 8.9 Hz, 2 H),
8.21 (d, J = 8.8 Hz, 2 H), 8.16 (s, 1 H, 0.5), 8.14 (d, J = 3.6 Hz, 1
H), 8.05 (s, 1 H), 7.90–7.85 (m, 2 H), 7.75–7.68 (m, 2 H), 7.20–7.12
(m, 2 H), 7.12–7.02 (m, 4 H), 6.96 (d, J = 8.5 Hz, 1 H), 6.89 (d, J
= 8.7 Hz, 1 H), 6.75 (d, J = 8.5 Hz, 1 H), 6.71 (d, J = 1.7 Hz, 2
H), 6.68 (d, J = 8.8 Hz, 1 H), 6.56 (dd, J = 13.8, 8.7 Hz, 2 H), 6.39
(d, J = 8.6 Hz, 1 H), 4.73 (t, J = 5.0 Hz, 1 H), 4.63 (t, J = 5.0 Hz,
1 H), 4.51 (s, 1 H), 4.47 (d, J = 1.5 Hz, 1 H), 4.45 (s, 1 H), 4.38 (t,
J = 5.2 Hz, 1 H), 4.21 (t, J = 5.1 Hz, 1 H), 2.44–2.32 (m, 2 H),
0.83 (td, J = 10.5, 7.4 Hz, 3 H) ppm. 13C NMR (125 MHz, [D6]-
DMSO): δ = 156.46, 156.09,155.62, 155.23, 148.34, 143.71, 143.59,
143.54, 142.16, 142.10, 140.09, 139.84, 137.73, 136.83, 136.27,
136.03, 133.81, 133.50, 131.41, 131.34, 130.28, 130.14, 130.09,
129.86, 129.34, 127.86, 127.81, 127.03, 125.96, 125.91, 124.76,
124.59, 124.48, 124.41, 124.21, 114.94, 114.28, 114.17, 113.38,
66.02, 65.79, 62.69, 62.65, 62.49, 62.47, 49.02, 48.93, 44.58, 28.53,
28.43, 13.37 ppm.
Ral-Ant: 30 mg, 73%, Rf = 0.65 (CH2Cl2/MeOH, 9:1), HRMS
(ESI, negative): m/z = calcd. 757.2239 (for [C44H33N6O5S]–), found
757.2248. 1H NMR (500 MHz, [D6]DMSO): δ = 9.78 (s, 1 H), 9.73
(s, 1 H), 8.72 (s, 1 H), 8.59 (dd, J = 8.9, 0.5 Hz, 2 H), 8.15 (d, J =
8.4 Hz, 2 H), 8.07 (s, 1 H), 7.92 (s, 1 H), 7.63 (m, 4 H), 7.59–7.47
(m, 2 H), 7.34 (d, J = 2.4 Hz, 1 H), 7.23 (d, J = 8.7 Hz, 1 H), 7.15
(d, J = 8.7 Hz, 2 H), 6.88 (d, J = 9.0 Hz, 2 H), 6.84 (dd, J = 8.8,
2.3 Hz, 1 H), 6.68–6.64 (d, J = 8.7 Hz, 2 H), 6.63 (s, 2 H), 4.69 (t,
J = 5.1 Hz, 2 H), 4.46 (s, 1 H), 4.43 (s, 1 H), 4.39 (t, J = 5.1 Hz, 2
H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 192.43, 61.89,
157.71, 155.31, 143.52, 140.37, 131.65, 130.91, 130.17, 129.57,
128.98, 126.99, 125.62, 125.24, 124.33, 123.86, 123.62, 123.52,
123.18, 115.60, 114.44, 107.01, 62.40, 48.58, 45.32 ppm.
Ral-Pyr: 22 mg, 80%, Rf = 0.59 (CH2Cl2/MeOH, 9:1), HRMS
(ESI, negative): m/z = calcd. 781.2239 (for [C46H33N6O5S]–), found
781.2237. 1H NMR (500 MHz, [D6]DMSO): δ = 9.22 (s, 1 H), 9.17
(s, 1 H), 7.97 (d, J = 9.25 Hz, 1 H), 7.83–7.69 (m, 4 H), 7.67–7.58
(m, 3 H), 7.54 (dd, J = 8.8, 6.4 Hz, 2 H), 7.46 (d, J = 7.9 Hz, 1 H),
7.07 (d, J = 8.9 Hz, 2 H), 6.78 (d, J = 2.3 Hz, 1 H), 6.67 (d, J =
8.8 Hz, 1 H), 6.59 (d, J = 8.7 Hz, 2 H), 6.32 (d, J = 9.0 Hz, 2 H),
6.28 (dd, J = 8.8, 2.3 Hz, 1 H), 6.10 (d, J = 8.7 Hz, 2 H), 5.80 (s,
2 H), 4.13 (t, J = 5.1 Hz, 2 H), 3.97–3.92 (m, 4 H), 3.83 (t, J =
5.1 Hz, 2 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 192.42,
161.89, 155.31, 143.83, 143.56, 139.11, 132.12, 131.66, 130.92,
130.60, 130.04, 129.58, 128.31, 128.18, 127.70, 127.50, 127.16,
126.40, 125.61, 125.45, 124.94, 124.12, 123.92, 123.63, 123.18,
122.65, 115.60, 114.44, 62.50, 50.70, 48.58 ppm.
OHT-Ant: 2.9 mg (E/Z-mixture, 1:1), 50%, Rf = 0.63 (CH2Cl2/
MeOH, 9:1), MS (ESI, positive): m/z = calcd. 713.3240 (for
1
[C45H41N6O3]+), found 713.3. H NMR (500 MHz, [D6]DMSO): δ
= 9.35 (s, 1 H), 9.10 (s, 1 H), 8.7 (s, 1 H), 8.53 (dd, J = 8.8, 0.5 Hz,
2 H), 8.09 (d, J = 8.3 Hz, 2 H), 8.05 (s, 0.5 H), 7.97 (s, 0.5 H), 7.86
(d, J = 7.7 Hz, 1 H), 7.60–7.54 (m, 2 H), 7.52–7.46 (m, 2 H), 7.14–
7.05 (m, 2 H), 7.01 (ddd, J = 14.3, 10.5, 8.0 Hz, 4 H), 6.89 (d, J =
8.5 Hz, 1 H), 6.82 (d, J = 8.7 Hz, 1 H), 6.67 (d, J = 8.6 Hz, 1 H),
6.61 (d, J = 8.9 Hz, 1 H), 6.56 (d, J = 1.7 Hz, 2 H), 6.50 (td, J =
16.4, 6.3 Hz, 2 H), 6.32 (d, J = 8.7 Hz, 1 H), 4.65 (t, J = 5.0 Hz, 1
H), 4.55 (t, J = 5.0 Hz, 1 H), 4.42 (s, 1 H), 4.38 (d, J = 1.6 Hz, 2
H), 4.35 (s, 1 H), 4.33–4.28 (m, 1 H), 4.14 (t, J = 5.1 Hz, 1 H),
2.38–2.25 (m, 2 H), 0.76 (td, J = 10.4, 7.3 Hz) ppm.
General Procedure for the Synthesis of SERM-MiDye Conjugates:
To a solution of a MiDye-COOH (10 μmol) and TSTU (3.9 mg,
13 μmol) in a DMF/water (7:3) mixture (50–100 μL) was added
diisopropylethylamine (4.9 μL, 30 μmol). The mixture was agitated
at 300 rpm for 30 min. After consumption of the MiDye-COOH
(typically 30–100 min), a solution of the SERM-amine (15 μmol)
in DMF (10–30 μL) was added. The mixture was agitated for at
least 30 min until ESI-MS showed completion of the reaction. The
reaction was quenched by addition of acetic acid (2.4 μL). The mix-
ture was diluted with acetonitrile/water (9:1) containing 0.1% TFA
(800 μL) and filtered through a syringe filter (PTFE, 0.45 μm). The
filtrate was purified by preparative HPLC (Nucleodur Gravity C18
A 5μ VP 250/21, pore size 210 Å) and by using solvents A (98.9%
water, 1% acetonitrile, 0.1% TFA) and B (98.9% acetonitrile, 1%
water, 0.1% TFA) in a linear gradient (3% BǞ100% B in 30 min)
at 15 mLmin-1 flow rate. The product containing fractions were
lyophylized and stored at 4 °C under exclusion of light.
OHT-Pyr: 16 mg (E/Z-mixture, 1:1), 63%, Rf = 0.62 (CH2Cl2/
MeOH, 9:1), MS (ESI, positive): m/z = calcd. 737.3235 (for
1
[C47H41N6O3]+), found 737.3. H NMR (500 MHz, [D6]DMSO): δ
= 9.44 (s, 1 H), 9.18 (s, 1 H), 8.54 (dd, J = 9.3, 1.4 Hz, 1 H), 8.37–
8.28 (m, 4 H), 8.25–8.15 (m, 3.5 H), 8.11 (dt, J = 7.8, 1.0 Hz, 1 H),
8.08 (s, 0.5 H), 8.03 (dd, J = 7.9, 1.9 Hz, 1 H), 7.15 (td, J = 24.6,
7.4 Hz, 2 H), 7.11–7.04 (m, 4 H), 6.96 (d, J = 8.5 Hz, 1 H), 6.89
(d, J = 8.8 Hz, 1 H), 6.76 (d, J = 8.5 Hz, 1 H), 6.68 (d, J = 8.8 Hz,
1 H), 6.61–6.50 (m, 2 H), 6.41 (d, J = 8.6 Hz, 1 H), 6.4 (d, J =
1.53 Hz, 2 H), 4.74 (t, J = 5.1 Hz, 1 H), 4.64 (t, J = 5.1 Hz, 1 H),
4.55 (d, J = 3.4 Hz, 2 H), 4.52 (s, 2 H), 4.38 (t, J = 5.1 Hz, 1 H),
4.21 (t, J = 5.1 Hz, 1 H), 2.38 (qd, J = 26.1, 7.4 Hz, 2 H), 0.83 (td,
J = 13.8, 7.4 Hz, 3 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ
= 156.36, 155.10, 155.52, 155.13, 143.85, 143.55, 143.49, 142.06,
142.00, 139.98, 139.73, 137.63, 136.17, 135.93, 133.71, 133.41,
131.30, 131.23, 130.91, 130.60, 130.05, 130.02, 129.99, 129.23,
128.96, 128.31, 128.17, 127.74, 127.69, 127.47, 127.14, 126.38,
125.84, 125.79, 125.60, 125.44, 124.92, 124.40, 124.33, 124.14,
123.93, 123.60, 122.63, 114.85, 114.18, 114.07, 113.28, 65.92, 65.68,
62.56, 62.52, 54.78, 50.71, 48.93, 48.83, 28.42, 28.32, 13.25 ppm.
OHT-DMIDCC: 1.3 mg, 57%. MS (ESI, positive): m/z = calcd.
769.4243 (for [C52H55N3O3]+), found 769.4, tR = 18.75 min (3–95%
B in 20 min).
Ral-DMIDCC: 7.5 mg, 70%. HRMS (ESI, positive): m/z = calcd.
1
Ral-Acr: 16 mg, 58%, Rf = 0.63 (CH2Cl2/MeOH, 9:1), HRMS 815.3382 (for [C51H49N3O5S]+), found 815.3. H NMR (500 MHz,
(ESI, negative): m/z = calcd. 758.2191 (for [C47H41N6O3]+), found
[D6]DMSO): δ = 9.79 (m, 2 H), 8.67 (t, J = 5.4 Hz, 1 H), 8.36 (m,
758.2195. H NMR (500 MHz, [D6]DMSO): δ = 9.79 (s, 1 H) 9.73 2 H), 8.05 (d, J = 1.5 Hz, 1 H), 7.90 (dd, J = 1.6, J = 8.4 Hz, 1
1
(s, 1 H), 8.66 (d, J = 8.8 Hz, 2 H), 8.21 (d, J = 8.7 Hz, 2 H), 8.14
H), 7.68 (m, 3 H), 7.47 (m, 2 H), 7.34 (m, 3 H), 7.24 (d, J = 8.7 Hz,
(s, 1 H), 8.09 (s, 1 H), 7.91–7.83 (m, 2 H), 7.75–7.68 (m, 2 H), 7.64 1 H), 7.19 (d, J = 8.7 Hz, 2 H), 6.98 (d, J = 9.0 Hz, 2 H), 6.85 (dd,
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Job/Unit: O43489
/KAP1
Date: 19-02-15 17:46:33
Pages: 11
O. Seitz et al.
J = 2.3, 8.7 Hz, 1 H), 6.69 (d, J = 8.7 Hz, 2 H), 6.59 (t, J = 12.4 Hz, 1036.4246. 1H NMR (500 MHz, [D6]DMSO): δ = 8.65 (m, 1 H),
FULL PAPER
1 H), 6.43 (d, J = 14.1 Hz, 1 H), 6.21 (d, J = 13.5 Hz, 1 H), 4.18
(t, J = 5.7 Hz, 1 H), 3.68 (s, 1 H), 3.65 (m, 5 H), 3.56 (s, 3 H), 1.70
7.98 (m, 2 H), 7.82 (m, 3 H), 7.64 (d, J = 7.3 Hz, 1 H), 7.55 (d, J
= 7.7 Hz, 1 H), 7.44 (t, J = 7.5 Hz, 1 H), 7.31 (t, J = 7.2 Hz, 2 H),
(d, J = 6.9 Hz, 1 H, 12 H) ppm. 13C NMR (125 MHz, [D6]DMSO): 7.17 (m, 2 H), 7.09 (m,4 H), 6.97 (d, J = 8.5 Hz, 2 H, 2 H), 6.73
δ = 192.48, 174.86, 171.71, 165.92, 162.66, 157.73, 155.33, 154.96, (dd, J = 8.7, J = 16.0 Hz, 2 H), 6.61 (m, 3 H), 6.39 (d, J = 8.7 Hz,
153.07, 145.34, 142.47, 141.34, 140.36, 140.22, 139.13, 132.16,
131.75, 129.73, 129.58, 129.37, 128.36, 127.98, 125.68, 125.40,
123.67, 123.17, 122.29, 121.23, 115.62, 115.12, 114.43, 111.63,
1 H), 6.27 (d, J = 12.3 Hz, 1 H), 4.16 (m, 3 H), 3.97 (m, 3 H), 3.62
(m, 3 H), 2.55 (m, 4 H), 2.39 (m, 2 H), 1.6–1.8 (m, 20 H), 0.84 (dt,
J = 2.1, J = 7.3 Hz, 1 H) ppm. 13C NMR (125 MHz, [D6]DMSO):
109.69, 107.03, 104.83, 102.63, 66.39, 66.25, 49.27, 48.00, 31.41, δ = 165.91, 160.37, 156.18, 155.99, 142.09, 140.04, 139.84, 137.75,
30.79, 27.08, 26.67 ppm.
135.52, 133.78, 131.35, 131.26, 130.07, 130.01, 129.27, 128.52,
127.99, 127.79, 127.72, 125.88, 122.37, 114.85, 114.18, 114.04,
113.24, 65.72, 50.71, 50.53, 28.44, 27.34, 26.80, 22.36, 22.32,
13.29 ppm.
Ral-IDCC: 7.1 mg, 67%. HRMS (ESI, negative): m/z = calcd.
1056.3239 (for [C57H58N3O11S3]–), found 1056.3237. 1H NMR
(500 MHz, [D6]DMSO): δ = 9.77 (br. s, 1 H, OH), 8.64 (t, J =
5.4 Hz, 1 H, NH), 8.4–8.28 (m, 2 H), 8.02 (d, J = 1.3 Hz, 1 H), Ral-ITCC: 11.5 mg, 71%, HRMS (ESI, negative): m/z = calcd.
7.86 (dd, J = 1.5, J = 8.4 Hz, 1 H), 7.67 (m, 3 H), 7.51 (d, J = 1082.3395 (for [C59H60N3O11S3]–), found 1082.3396. 1H NMR
8.1 Hz, 1 H) ppm 7.42 (m, 2 H), 7.33 (d, J = 2.3 Hz, 1 H), 7.29 (t, (500 MHz, [D6]DMSO): δ = 9.74 (br. s, 2 H), 8.85 (m, 0.5 H), 8.60
J = 7.5 Hz, 1 H), 7.23 (d, J = 8.7 Hz, 1 H), 7.17 (d, J = 8.7 Hz, 2 (t, J = 5.3 Hz, 1 H), 7.98 (m, 2 H), 7.83 (dd, J = 1.3, J = 8.4 Hz,
H), 6.97 (d, J = 9.0 Hz, 2 H), 6.85 (dd, J = 2.2, J = 8.7 Hz, 1 H),
6.68 (d, J = 8.7 Hz, 2 H), 6.63 (t, J = 12.4 Hz, 1 H), 6.51 (d, J =
14.0 Hz, 1 H), 6.31 (d, J = 13.5 Hz, 1 H), 4.11 (m, 6 H), 3.63 (dd,
1 H), 7.76 (m, 2 H), 7.65 (dd, J = 8.2, J = 14.2 Hz, 2 H), 7.55 (d,
J = 8.0 Hz, 1 H), 7.44 (t, J = 8.1 Hz, 1 H), 7.32 (dd, J = 4.5, J =
11.6 Hz, 1 H), 7.23 (d, J = 8.8 Hz, 1 H), 7.17 (d, J = 8.7 Hz, 2 H),
J = 5.3, J = 10.6 Hz, 2 H), 2.55 (m, 4 H), 1.76 (m, 20 H) ppm. 13C 6.97 (d, J = 9.0 Hz, 2 H), 6.85 (dd, J = 2.3, J = 8.8 Hz, 1 H), 6.68
NMR (125 MHz, [D6]DMSO): δ = 192.46, 174.16, 171.08, 165.99,
162.67, 157.73, 155.31, 155.01, 153.22, 144.66, 141.70, 141.41,
140.48, 140.18, 139.11, 132.15, 131.72, 129.69, 129.55, 129.48,
128.42, 127.96, 126.21, 125.25, 123.65, 123.16, 122.34, 121.30,
115.61, 115.09, 114.42, 111.77, 109.94, 107.01, 104.79, 102.85,
66.36, 50.62, 50.52, 49.24, 48.06, 43.65, 43.04, 27.20, 26.87, 26.03,
25.73, 22.27 ppm.
(d, J = 8.7 Hz, 1 H, 2 H), 6.60 (m, 3 H), 6.27 (d, J = 12.9 Hz, 1
H), 4.16 (m, 4 H), 3.98 (m, 2 H), 3.63 (m, 2 H), 2.55 (m, 4 H), 1.77
(m, 8 H), 1.65 (s, 6 H), 1.61 (s, 6 H) ppm. 13C NMR (125 MHz,
[D6]DMSO): δ = 192.46, 166.02, 162.68, 157.73, 155.31, 141.68,
140.18, 140.02, 139.11, 132.15, 13.72, 129.68, 129.56, 128.53,
127.97, 123.64, 123.17, 122.36, 115.61, 115.09, 114.43, 107.01,
66.38, 50.53, 27.34, 26.79, 22.34 ppm.
OHT-DMITCC: 6.6 mg (E/Z-mixture, 1:1), 55%, MS (ESI, posi-
Binding Assays: Measurements of relative binding affinity to ERα:
The relative binding affinities were determined by using the Hi-
thunter Estrogen Assay Kit (DiscoverX, http://www.discoverx.com)
and human recombinant estrogen receptor α (Calbiochem). In this
1
tive): m/z = calcd. 795.4389 (for [C54H57N3O3]+), found 795.4. H
NMR (500 MHz, [D6]DMSO): δ = 9.15 (s, 1 H), 8.63 (t, J = 5.4 Hz,
1 H), 7.99 (m, 2 H), 7.90 (dd, J = 1.6, J = 8.4 Hz, 1 H), 7.76 (t, J
= 12.8 Hz, 2 H), 7.65 (d, J = 7.3 Hz, 1 H), 7.48 (m, 2 H), 7.34 (t, competition assay, a steroid-hormone-conjugate is attached to an
J = 7.6 Hz, 1 H), 7.28 (d, J = 8.4 Hz, 1 H), 7.17 (t, J = 7.7 Hz, 2
H), 7.09 (m, 5 H), 6.97 (d, J = 8.7 Hz, 2 H), 6.73 (dd, J = 8.7, J =
enzyme fragment (A). In the presence of estrogen-receptor this con-
jugate is bound. The conjugate is not able to bind to a second
13.8 Hz, 1 H), 6.59 (m, 5 H), 6.39 (d, J = 8.6 Hz, 2 H), 6.14 (d, J enzyme fragment (B). In the presence of estradiol the steroid hor-
= 13.3 Hz, 1 H), 4.15 (t, J = 5.7 Hz, 2 H), 3.67 (m, 6 H), 3.48 (m,3 mone-conjugate (A) is displaced and liberated into solution. Free
H), 2.38 (q, J = 7.4 Hz, 2 H), 1.64 (m, 12 H), 0.84 (t, J = 7.4 Hz, unbound steroid-hormone conjugate (A) binds B and forms an
3 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 165.88, 157.00,
155.12, 145.62, 142.41, 142.10, 141.47, 139.86, 139.66, 137.73,
135.78, 133.48, 131.34, 131.23, 130.06, 129.98, 129.24, 128.43,
127.98, 127.77, 127.72, 125.80, 122.30, 121.12, 114.84, 114.17,
114.01, 113.21, 111.97, 109.01, 65.96, 49.36, 47.31, 31.68, 30.53,
28.34, 27.23, 26.53, 13.30 ppm.
active β-galactosidase enzyme which cleaves a substrate to generate
a chemoluminescent signal. For the measurements of the relative
binding affinity (RBA), the SERM conjugates were dissolved in
DMSO as described by the manufacturer protocol.
Prior to the measurements, the SERM conjugates were diluted with
DMSO to prepare 9 serial 1:3 or 1:2 dilutions in Eppendorf tubes.
An aliquot (1 μL) of the SERM-containing solution was transfer-
Ral-DMITCC: 7.8 mg, 62%, MS (ESI, positive): m/z = calcd.
1
841.3538 (for [C53H51N3O5S]+), found 841.3. H NMR (500 MHz, red to a well of a 384 microtiter plate (Greiner Bio one 120 μL,
[D6]DMSO): δ = 9.79 (s, 1 H), 9.74 (s, 1 H), 8.61 (t, J = 5.4 Hz, 1 white) and 25 μL of a receptor/ED-mix (ED = steroid-hormone
H), 7.98 (m, 2 H), 7.86 (dd, J = 1.6, J = 8.4 Hz, 1 H), 7.74 (t, J =
12.7 Hz, 1 H, m, 2 H), 7.66 (dd, J = 8.0, J = 10.5 Hz, 1 H), 7.48
(m, 2 H), 7.34 (m, 2 H), 7.26 (d, J = 8.4 Hz, 1 H), 7.23 (d, J =
conjugate in which the steroid-hormone is recognized by the steroid
hormone receptor) was added. The mixture was incubated for
90 min at ambient temperature. Afterwards 10 μL EA (solution of
8.7 Hz, 1 H), 7.17 (d, J = 8.7 Hz, 2 H), 6.97 (d, J = 9.0 Hz, 2 H), an inactive enzyme which is activated upon binding of free ED)
6.84 (dd, J = 2.2, J = 8.7 Hz, 1 H), 6.68 (d, J = 8.7 Hz, 2 H), 6.55 and 10 μL substrate (enzyme-catalyzed cleavage triggers chemilu-
(m, 3 H), 6.13 (d, J = 13.2 Hz, 1 H), 4.16 (t, J = 5.7 Hz, 2 H), 3.70 minescence) were added. The readout was performed on plate
(s, 3 H), 3.62 (dd, J = 5.5, J = 11.1 Hz, 2 H), 3.47 (s, 3 H), 1.65 (s,
reader (Victor X5 or Polarstar Optima, BMG Labtech) after
6 H), 1.61 (s, 6 H) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 90 min.
192.48, 165.95, 162.67, 157.73, 155.33, 147.56, 145.68, 142.43,
Molecular Dynamics Analysis: For parametrization of the struc-
141.51, 140.21, 139.87, 139.14, 132.16, 131.75, 129.73, 129.59,
128.63, 128.46, 127.99, 125.85, 125.82, 123.68, 123.18, 122.33,
121.13, 115.63, 115.12, 114.43, 112.02, 109.01, 107.04, 66.42, 49.40,
47.30, 31.71, 30.53, 27.24, 26.55 ppm.
tures the Amber force field AMBER99SB was used.[11] To generate
the topology files of the ligands the Python based tool Acpype
was used.[12] For the molecular dynamic simulations, which were
performed with GROMACS 4.6.5[13] and the AMBER99SB force
OHT-ITTC: 7.7 mg (E/Z-mixture, 1:1), 52%, HRMS (ESI, nega- field, an octahedral simulation box with a box distance of 0.6 nm
tive): m/z
=
calcd. 1036.4246 (for [C60H66N3O9S2]–), found
was filled with explicit water using the TIP4P-Ew water model.[14]
8
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High Affinity Fluorescent Estrogen Receptor Ligands
To neutralize charges the appropriate kind and amount of ions
were added. The calculations were started with an energy minimi-
zation that was performed with steepest descent. This generated an
input for further computations, which were carried out at a con-
stant temperature of 310 K and constant pressure using Berendsen
weak coupling. For the complexes and for the ligands in solvent
a molecular simulation was performed. The bound and unbound
systems were used to extract interaction energies of the compounds
and thereby to predict binding affinities of the two different ligand–
receptor complexes.
the cells were washed 3 times with PBS buffer. After this step the
cells were covered with fluorescence mounting medium (Dako
S3023). Fixed cells were kept at 4 °C until analyzed. Fluorescence
microscopy was performed by using a Zeiss LSM5 Exciter micro-
scope (Carl Zeiss, Germany). Data was analyzed with Zen software
(Carl Zeiss, Germany).
Supporting Information (see footnote on the first page of this arti-
cle): General methods and materials, synthesis of tag azides, optical
spectroscopy, competitive binding curves, NMR spectra.
Fluorescence Microscopy
Acknowledgments
Cell Culture: MCF-7 and HEK cells were grown using DMEM
medium containing 10% fetal calf serum (FCS), stable glutamine
w/o sodium pyruvate, 10 μg/L human insulin and penicillin/strepto-
mycin (Pan Biotech). SKBR-3 cells were grown using McCoyЈs 5a
medium modified with 10% fetal calf serum (FCS) and penicillin/
streptomycin (Pan Biotech). A2780 cells were grown using RPMI-
medium containing 10% FCS fetal calf serum (FCS), 2 mm
glutamine and penicillin/streptomycin (Pan Biotech).
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) (SFB 765).
[1] a) K. Dahlman-Wright, V. Cavailles, S. A. Fuqua, V. C. Jordan,
J. A. Katzenellenbogen, K. S. Korach, A. Maggi, M. Muram-
atsu, M. G. Parker, J. A. Gustafsson, Pharmacol. Rev. 2006, 58,
773–781; b) D. J. Mangelsdorf, C. Thummel, M. Beato, P.
Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M.
Mark, P. Chambon, R. M. Evans, Cell 1995, 83, 835–839; c)
J. M. Olefsky, J. Biol. Chem. 2001, 276, 36863–36864.
[2] a) C. M. Revankar, D. F. Cimino, L. A. Sklar, J. B. Arterburn,
E. R. Prossnitz, Science 2005, 307, 1625–1630; b) S. H. Kim,
J. R. Gunther, J. A. Katzenellenbogen, J. Am. Chem. Soc. 2010,
132, 4685–4692; c) F. J. Cespedes-Guirao, A. B. Ropero, E.
Font-Sanchis, A. Nadal, F. Fernandez-Lazaro, A. Sastre-San-
tos, Chem. Commun. 2011, 47, 8307–8309.
Comparison of Unconjugated IDCC-dye to Conjugated Ral-IDCC:
5ϫ105 MCF-7 cells were seeded out in 96 well plates in 100 μL/
well culture medium [DMEM medium with 10% fetal calf serum
(FCS), stable glutamine w/o sodium pyruvate, 10 μg/L human insu-
lin and penicillin/streptomycin (Pan Biotech)] and incubated for
24 h at 37 °C and 5% CO2. Afterwards the cells were treated with
2 μL of a stock solution (5 μm) of IDCC-COOH or Ral-IDCC in
biological grade DMSO (final concentration: 100 nM). After
30 min incubation cells were washed twice with PBS buffer and
images were acquired without fixation using a Zeiss AXIO observer
microscope. For IDCC-COOH a higher excitation energy was used.
[3] E. L. Rickert, S. Oriana, C. Hartman-Frey, X. H. Long, T. T.
Webb, K. P. Nephew, R. V. Weatherman, Bioconjugate Chem.
2010, 21, 903–910.
[4] S. H. Kim, J. R. Gunther, J. A. Katzenellenbogen, Org. Lett.
2008, 10, 4931–4934.
Comparison Between Ral-IDCC Stains of MCF-7 and HEK Cells:
One day before measurement 250.000 cells were seeded out by
using 3 mL culture media in a 6-well plate on coverslips. On the
next day Ral-IDCC in DMSO was added to each well to yield a
final concentration of 5 nM Ral-IDCC and 1% DMSO. Cells were
incubated for 20 min and then rapidly washed once with pre-
warmed DMEM medium which contained 10% fetal calf serum
(FCS), stable glutamine w/o sodium pyruvate, 10 μg/L human
insulin and penicillin/streptomycin (Pan Biotech). The cells were
incubated for 20 min with culture media at 37 °C, 5% CO2 and
washed twice with PBS buffer. Afterwards cells were counterstained
with 1 mL of a 100 nM Dapi solution in PBS buffer for 5 min.
Subsequently, the coverslips were withdrawn. The cells attached
were washed twice with PBS buffer and fixed upon 8 min treatment
with 3.7% formaldehyde solution. The coverslips were washed with
PBS buffer and covered with 50% glycerol in PBS buffer.
[5] a) K. J. Hwang, K. E. Carlson, G. M. Anstead, J. A. Katzenel-
lenbogen, Biochemistry 1992, 31, 11536–11545; b) K. K. W. Lo,
T. K. M. Lee, J. S. Y. Lau, W. L. Poon, S. H. Cheng, Inorg.
Chem. 2008, 47, 200–208; c) M. Salman, B. R. Reddy, P.
Delgado, P. L. Stotter, L. C. Fulcher, G. C. Chamness, Steroids
1991, 56, 375–387; d) K. Ohno, T. Fukushima, T. Santa, N.
Waizumi, H. Tokuyama, M. Maeda, K. Imai, Anal. Chem.
2002, 74, 4391–4396.
[6] F. Abendroth, A. Bujotzek, M. Shan, R. Haag, M. Weber, O.
Seitz, Angew. Chem. Int. Ed. 2011, 50, 8592–8596.
[7] a) M. Shan, K. E. Carlson, A. Bujotzek, A. Wellner, R. Gust,
M. Weber, J. A. Katzenellenbogen, R. Haag, ACS Chem. Biol.
2013, 8, 707–715; b) M. Shan, A. Bujotzek, F. Abendroth, A.
Wellner, R. Gust, O. Seitz, M. Weber, R. Haag, ChemBioChem
2011, 12, 2587–2598.
[8] a) D. J. Kojetin, T. P. Burris, E. V. Jensen, S. A. Khan, Endocr.-
Relat. Cancer 2008, 15, 851–870; b) Y. Wang, N. Y. Chirgadze,
S. L. Briggs, S. Khan, E. V. Jensen, T. P. Burris, Proc. Natl.
Acad. Sci. USA 2006, 103, 9908–9911.
[9] a) A. K. Shiau, D. Barstad, P. M. Loria, L. Cheng, P. J.
Kushner, D. A. Agard, G. L. Greene, Cell 1998, 95, 927–937;
b) A. M. Brzozowski, A. C. W. Pike, Z. Dauter, R. E. Hubbard,
T. Bonn, O. Engstrom, L. Ohman, G. L. Greene, J. A. Gustafs-
son, M. Carlquist, Nature 1997, 389, 753–758; c) A. C. W. Pike,
A. M. Brzozowski, R. E. Hubbard, T. Bonn, A. G. Thorsell, O.
Engstrom, J. Ljunggren, J. K. Gustafsson, M. Carlquist,
EMBO J. 1999, 18, 4608–4618.
[10] V. Durmaz, S. Schmidt, P. Sabri, C. Piechotta, M. Weber, J.
Chem. Inf. Model. 2013, 53, 2681–2688.
[11] D. A. Case, V. Babin, J. T. Berryman, R. M. Betz, Q. Cai, T. E.
Cerutti, T. E. Cheatham, T. E. Darden, R. E. Duke, H. Gohlke,
A. W. Goetz, S. Gusarov, N. Homeyer, P. Janowski, J. Kaus, I.
Kolossvary, A. Kovalenko, T. S. Lee, S. LeGrand, T. Luchko,
R. Luo, B. Madej, K. M. Merz, F. Paesani, D. R. Roe, A. Roit-
Competition Between Ral-IDCC and E2 in MCF-7 and SKBR-3
Cells: The adherent cells were plated in 24-well-culture plates on
glass coverslips (Sigma) (2ϫ10–5cells/well) with 0.5 mL pre-
warmed culture medium the day before measurements were com-
menced. On the day of measurement media was exchanged by
culture medium containing 1% FCS. Ral-IDCC was dissolved in
biological grade DMSO (10 μm). Aliquots were added to give a
final concentration of 100 nM per well. After 10 min of incubation
at 37 °C and 5% CO2 cells were washed 3 times with pre-warmed
PBS buffer. Afterwards the cells were incubated for 30 min in cul-
ture medium containing 1% FCS with or without 17-β-estradiol
(10 μm final concentration). Subsequently, the cells were washed 3-
times with pre-warmed PBS buffer and fixed upon treatment with
4% paraformaldehyde on ice for 10 min. The coverslips were rinsed
with PBS buffer and a 1:2000 dilution of 2.5 μg/mL Dapi (Roche)
in water was added. After 10 min incubation at room temperature
Eur. J. Org. Chem. 0000, 0–0
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O. Seitz et al.
FULL PAPER
berg, C. Sagui, R. Salomon-Ferrer, G. Seabra, C. L. Sim-
merling, W. Smith, J. Swails, R. C. Walker, J. Wang, R. M.
Wolf, X. Wu, P. A. Kollmann, 2014, AMBER 14, University of [14] H. W. Horn, W. C. Swope, J. W. Pitera, J. D. Madura, T. J.
California, San Francisco.
[13] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem.
Theory Comput. 2008, 4, 435–447.
Dick, G. L. Hura, T. Head-Gordon, J. Chem. Phys. 2004, 120,
9665–9678.
[12] A. W. Sousa da Silva, W. F. Vranken, BMC Research Notes
2012, 5, 367.
Received: November 18, 2014
Published Online: ᭿
10
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O43489
/KAP1
Date: 19-02-15 17:46:33
Pages: 11
High Affinity Fluorescent Estrogen Receptor Ligands
Protein–Ligand Interactions
Dual click reactions have facilitated hydro-
phobic tagging of selective estrogen recep-
tor modulators (SERMs). Raloxifene con-
jugation with fluorescent dyes afforded
fluorescent ER with affinities superior to
estradiol. These stains accumulate in ER
expressing cells at low nanomolar concen-
trations and molecular dynamics simu-
lations suggest dye–ER associations via
helix 12 contacts.
F. Abendroth, M. Solleder, D. Mangoldt,
P. Welker, K. Licha, M. Weber,
O. Seitz* ......................................... 1–11
High Affinity Fluorescent Ligands for the
Estrogen Receptor
Keywords: Click chemistry / Hormones /
Fluorescence / Fluorescent probes / Recep-
tor–ligand interactions / Molecular simu-
lation / Cyanines
Eur. J. Org. Chem. 0000, 0–0
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11