Development of a time-resolved fluorescence probe for evaluation of competitive binding to the cholecystokinin 2 receptor

Article abstract of DOI:10.1016/j.bmc.2015.02.028

The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH₂ (Eu-DTPA-PEGO-CCK4), a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe was prepared by solid phase synthesis. A K_d value of 17 ± 2 nM was determined by means of saturation binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties, and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-throughput screening of compounds in development targeted to cholecystokinin receptors.

Full text of DOI:10.1016/j.bmc.2015.02.028

Bioorganic & Medicinal Chemistry 23 (2015) 1841–1848
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a time-resolved fluorescence probe for evaluation
of competitive binding to the cholecystokinin 2 receptor
N. G. R. Dayan Elshan a, Thanuja Jayasundera b, Craig S. Weber b, Ronald M. Lynch b,c, Eugene A. Mash a,
⇑
a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0041, USA
b Department of Physiology, University of Arizona, Tucson, AZ 85724-5051, USA
c The Bio5 Institute, University of Arizona, Tucson, AZ 85721-0240, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, characterization, and use of Eu-DTPA-PEGO-Trp-Nle-Asp-Phe-NH2 (Eu-DTPA-PEGO-CCK4),
a luminescent probe targeted to cholecystokinin 2 receptor (CCK2R, aka CCKBR), are described. The probe
was prepared by solid phase synthesis. A Kd value of 17 2 nM was determined by means of saturation
binding assays using HEK-293 cells that overexpress CCK2R. The probe was then used in competitive
binding assays against Ac-CCK4 and three new trivalent CCK4 compounds. Repeatable and reproducible
binding assay results were obtained. Given its ease of synthesis, purification, receptor binding properties,
and utility in competitive binding assays, Eu-DTPA-PEGO-CCK4 could become a standard tool for high-
throughput screening of compounds in development targeted to cholecystokinin receptors.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 9 January 2015
Revised 6 February 2015
Accepted 16 February 2015
Available online 26 February 2015
Keywords:
Multivalent binding
Cholecystokinin 2 receptor
High-throughput screening
Time-resolved fluorescence
Dissociation-enhanced lanthanide
fluoroimmunoassay
1. Introduction
When developing a new series of compounds, rapid screening for
binding efficacy using a high-throughput competitive binding
The cholecystokinin 2 receptor (CCK2R, aka CCKBR) is a member
of the GPCR family expressed in several cancers, including
medullary thyroid carcinomas, small-cell lung cancers, gastroen-
teropancreatic neuroendocrine cancers, stromal ovarian cancers,
astrocytomas, and gastrointestinal stromal cancers.1 In many of
these cancers, CCK2R is overexpressed on malignant cell sur-
faces,2,3 providing an opportunity for targeted imaging and/or ther-
apy. One strategy for detecting overexpression is to employ
simultaneous binding of two or more weak ligands presented on
a single scaffold. Such multivalent molecules can selectively bind
with high avidity to cells overexpressing the targeted receptors.4,5
assay is a useful strategy for hit selection. By this method,
the binding efficacies of a series of unlabeled molecules may be
evaluated against a single labeled probe targeted to the same
receptor. While many radiolabelled ligands that bind to CCK recep-
tors have been reported,6–9 luminescent probes targeted to CCKRs
were only recently described. Probes bearing lanthanide chelates
have become popular due to their sensitivity and the avoidance
of radioactive materials.10–13 They are of special importance in
applications where background signal is a significant problem.
During the past decade several europium-diethylenetriaminepen-
taacetic acid (Eu-DTPA) chelates linked to peptide recognition
elements have been reported.10,13–16 These probes were used to
evaluate multivalent molecules targeted to melanocortin or
cholecystokinin receptors via dissociation-enhanced lanthanide
fluoroimmunoassay (DELFIA)-based ligand binding assays.17 The
functional readout of such assays comes from the time-resolved
fluorescence (TRF) generated by the europium ions. While TRF
probes 1 and 2 based on a CCK8 recognition element16 have previ-
ously been used to characterize ligand binding to CCK2R in com-
petitive binding assays,18–20 a redesign of these probes could
simplify and improve probe preparation and purification, possibly
enhance probe solubility and receptor binding, and provide an
additional tool for use in bioassays.
Abbreviations: CCK4, Trp-Nle-Asp-Phe-NH2; Cl-HOBt, 1-hydroxy-6-chloroben-
zotriazole; CuAAC, copper-catalyzed azide–alkyne cyclization; DELFIA, dissociation-
enhanced lanthanide fluoroimmunoassay; DIC, diisopropyl carbodiimide; DMEM,
Dulbecco’s Modified Eagle Medium; DTPA, diethylenetriaminepentaacetic acid; ESI,
electrospray ionization; ICR, ion cyclotron resonance; hCCK2R, human cholecys-
tokinin
2 receptor; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid;
hMC4R, human melanocortin 4 receptor; HOBt, 1-hydroxybenzotriazole; PEGO,
19-amino-5-oxo-3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid; TBTA, tris[(1-
benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TACP, tetrakis(acetonitrile)copper(I)
hexafluorophosphate; TRF, time-resolved fluorescence.
⇑
Corresponding author. Tel.: +1 520 621 6321; fax: +1 520 621 8407.
E-mail address: emash@email.arizona.edu (E.A. Mash).
http://dx.doi.org/10.1016/j.bmc.2015.02.028
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1842
N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
was used at 220 and 280 nm. A VWR SympHony™ pH meter
O
(Model SB20) equipped with a Ag/AgCl electrode was used for pH
measurements. ESI experiments were performed on a Bruker 9.4
T Apex-Qh hybrid Fourier transform ion-cyclotron resonance
(FT-ICR) instrument using standard ESI conditions. Samples were
dissolved in MeCN/water (1:1) or MeCN/MeOH (1:1) containing
O
N
Eu
Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2
N
N
HO
O
O
O
O
O
0.1% formic acid in a concentration range of 1–30 lM. HEK-293
O
1
cells engineered18 to overexpress both hCCK2R and hMC4R were
used to measure the affinity of the probe for binding to hCCK2R
by means of saturation binding assays. All compounds evaluated
in bioassays had purities of P95% as determined by HPLC. Unless
otherwise specified, all cell incubations were done in a Fisher
Scientific™ Isotemp™ CO2 incubator (Model 3530) maintained at
37 °C and 5% CO2 atmosphere. Europium-based TRF competitive
binding assays were employed to study the binding of all multiva-
lent constructs and controls. Centrifugations were performed using
a VWR Galaxy 7 microcentrifuge or a Fischer Scientific Model 59A
microcentrifuge. TRF was measured using a VICTOR™ X4 2030
Multilabel Reader (PerkinElmer) employing the standard Eu TRF
O
O
N
Eu
PEG9-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2
N
N
O
HO
O
O
O
O
O
2
In designing probes for characterization of multivalent binding,
we employ the minimum peptide sequence that gives an accept-
able level of receptor binding on the theory that a binding compe-
tition should involve recognition elements of similar binding
affinity. This strategy also minimizes the number of synthetic steps
required for probe assembly. In this manner we recently developed
a new and useful TRF probe for the evaluation of ligand binding to
human melanocortin 4 receptors (hMC4R) and established a robust
in vitro binding assay protocol.15,21 In the present work, we
describe a new TRF probe for high-throughput screening of
compounds for binding to CCK2R and extend the aforementioned
binding assay protocol to bioassays involving this receptor.
measurement settings (340 nm excitation, 400 ls delay, and
emission collection for 400 ls at 615 nm).
2.2. Solid-phase synthesis
2.2.1. Resin-bound protected CCK4 peptide 4
For the synthesis of probe 3 (see Scheme 1) and compounds 6,
10, 12, and 14 (see Schemes 2 and 3), resin-bound protected
a
CCK4 peptide 4 was synthesized manually via an N -Fmoc solid-
phase peptide synthesis strategy on Rink amide AM resin (200–
400 mesh, 0.68 mmol/g loading). Resin (1 g) was allowed to swell
in THF for 1 h in a polypropylene syringe equipped with a
polypropylene frit. THF was removed, a solution of 20% piperidine
in DMF (15 mL) was added, and the tube was shaken for 2 min.
This solution was removed, 20% piperidine in DMF (15 mL) was
again added, and the mixture was shaken for 18 min. After removal
of the solution, the resin was washed sequentially with DMF
(3 ꢀ 15 mL), DCM (3 ꢀ 15 mL), DMF (3 ꢀ 15 mL), 0.5 M HOBt in
DMF (15 mL), 0.5 M HOBt in DMF + one drop of 0.01 M bromophe-
nol blue solution in DMF (15 mL), DMF (2 ꢀ 15 mL), and DCM
(15 mL) in that order. Unless otherwise specified, all resin wash
steps in the solid phase peptide synthesis were done by shaking
the resin in contact with the wash solvent for 1 min. The amino
acid (3 equiv) to be coupled was activated by reaction in a glass
vial with 1-hydroxy-6-chlorobenzotriazole (Cl-HOBt, 3 equiv) and
diisopropyl carbodiimide (DIC, 6 equiv) in DMF (15 mL) over two
min. This solution was then added to the resin and the syringe
shaken for 1 h. The coupling solution was removed and the resin
was washed with DMF (3 ꢀ 15 mL), DCM (3 ꢀ 15 mL), and DMF
(3 ꢀ 15 mL). Free amine groups were capped by shaking the resin
with acetic anhydride/pyridine (1:1, 6 mL) for 20 min. The resin
was washed with DMF (3 ꢀ 15 mL), DCM (3 ꢀ 15 mL), and DMF
(3 ꢀ 15 mL). The coupling cycle was then repeated for each of the
remaining amino acids in the sequence. The Kaiser test22 was used
to determine coupling completion at each attachment step.
Following the four coupling cycles, resin-bound 4 was obtained
in its protected form.
2. Materials and methods
2.1. General
Commercial reagents were used as supplied unless otherwise
noted. Dichloromethane (DCM) and tetrahydrofuran (THF) were
dried by passage through activated alumina. Dimethylsulfoxide
(DMSO) and N,N-dimethylformamide (DMF) were dried by contact
with activated 4 Å molecular sieves, followed by distillation under
reduced pressure. Analytical thin-layer chromatography (TLC) was
carried out on pre-coated silica gel 60 F-254 plates with staining by
10% phosphomolybdic acid solution in ethanol or aqueous potassi-
um permanganate solution and heat. Column chromatography was
performed using silica gel 60 (200–400 mesh). Melting points were
recorded on an ElectrothermalÒ Mel-TempÒ apparatus (Model
1001) and are uncorrected. IR spectra were recorded on
a
Thermo Nicolet iS5 FT-IR Spectrophotometer using NaCl plates.
NMR spectra were recorded at 500 MHz for 1H NMR and at
125 MHz for 13C NMR on a Bruker DRX-500 NMR instrument.
Chemical shifts (d) are expressed in ppm and are internally refer-
enced to chloroform (7.26 ppm and 77.16 ppm for 1H and 13C
NMR, respectively). Reactions under microwave irradiation utilized
Biotage Initiator 2.0 microwave reactor. Preparative scale
reversed-phase HPLC was performed using
19 ꢀ 250 mm
Waters XBridge™ 10 m OBD™ C18 preparative HPLC column.
a
a
l
The flow rate was 10 mL/min and a dual channel UV detector
was used at 230 and 280 nm. Analytical HPLC was performed on
2.2.2. DTPA-PEGO-CCK4 (5)
a 3.0 ꢀ 150 mm Waters XBridge™ 3.5
lm C18 analytical HPLC col-
The PEGO linker was attached to a portion (ꢁ0.34 mmol) of
resin-bound peptide 4 by a conventional coupling cycle following
activation of Fmoc-PEGO-OH (Novabiochem 8510310001,
1.02 mmol in 2.04 mL of DCM) with Cl-HOBt (173 mg, 1.02 mmol)
and DIC (256 mg, 2.04 mmol) in DMF (5 mL). After 2 h, coupling
completion was ascertained by the Kaiser test, and the resin was
washed sequentially with DMF (3 ꢀ 8 mL), DCM (3 ꢀ 8 mL), and
DMF (3 ꢀ 8 mL).
umn. For the analysis of compound 3, a linear gradient of mobile
phase from 10% to 90% MeCN/triethylammonium acetate buffer
(0.1% v/v of triethylamine in HPLC grade water adjusted to
pH = 6.0 by the addition of acetic acid) was used over 30 min. For
all other compounds, a linear gradient of mobile phase from 10%
to 90% MeCN/water containing 0.1% TFA was used over 30 min.
The flow rate was 0.3 mL/min and a dual channel UV detector

N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
1843
Amino acid
coupling
H
N
H
N
1) PEGO Coupling
Fmoc
Fmoc-Trp(Boc)-Nle-Asp(OtBu)-Phe
2) DTPA dianhydride, HOBt,
DMSO
3) TFA, Thioanisole, TIPS, H2O
Rink Amide AM resin
(0.68 mmol/g)
4
O
O
O
OH
H
H
N
Eu
N
O
O
N
O
O
N
N
O
N
N
O
N
OH
OH
EuCl3
O
O
O
O
O
O
O
OH
HO
O
O
O
O
NH4OAc
Buffer
H
H
N
O
Trp-Nle-Asp-Phe-NH2
N
Trp-Nle-Asp-Phe-NH2
O
O
O
O
DTPA-PEGO-CCK4
Eu-DTPA-PEGO-CCK4
5
3
35%
73%
a
Scheme 1. Synthesis of the probe Eu-DTPA-PEGO-CCK4 (3) using an N -Fmoc solid-phase peptide synthesis strategy.
N
CCK4
CCK4
O
N
N
O
O
N3
CCK4
CCK4
N
10
N
N
N
N
O
N
N
TACP
N
2,6-lutidine
DMF
rt, 4 d
7
O
12%
N
N
N
N
N
N
O
O
N3
O
CCK4
12
CCK4
CCK4
O
N
O
O
O
TACP
TBTA
O
N
N
sodium ascorbate
DMF
11
O
microwave (100 oC), 4 h
8
CCK4
12%
Scheme 2. Synthesis of 7 and 8.
O
OR2
O
OR1
O
CCK4
4
14
R2O
OR2
R1O
OR1
TCPA
TBTA
sodium ascorbate
N
N
R1 = (CH2)6N3
N
O
2
(CH )
R =
2 6
O
DMF
13
O
CCK4
microwave (100 oC), 4 h
4
9
17%
Scheme 3. Synthesis of 9.

1844
N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
One half of the resin from above (ꢁ0.17 mmol) was placed in a
1728; 1H NMR (500 MHz, CDCl3) d 10.07 (s, 1H), 3.97 (s, 2H); 13C
NMR (125 MHz, CDCl3) d 174.27, 50.11.
syringe reactor and 20% piperidine in DMF (4 mL) was added. The
syringe was shaken for 2 min and the solution removed. Additional
20% piperidine in DMF (4 mL) was added and the syringe shaken
for 18 min. The solution was removed and the resin washed thor-
oughly with dry DMSO (9 ꢀ 4 mL). DTPA dianhydride (620 mg,
1.7 mmol, 10 equiv) and HOBt monohydrate (520 mg, 3.4 mmol,
20 equiv) were placed in a capped vial with dry DMSO (5 mL).
This suspension was heated at 70 °C for 5 min. The suspension
cleared, the solution was stirred for 15 min at rt, and was then tak-
en up into the syringe reactor. The mixture was shaken for 1 h and
the resin washed with DMSO (2 ꢀ 4 mL), THF (2 ꢀ 4 mL), 20%
aqueous THF (4 mL), 5% diisopropylethylamine in THF (4 mL),
THF (3 ꢀ 4 mL), and DCM (2 ꢀ 4 mL) with five min of shaking for
each wash.
Using the coupling procedures described previously, compound
10 was prepared from azidoacetic acid (0.21 g, 2.04 mmol, 3 equiv)
and resin-bound 4 (ꢁ0.68 mmol). Cleavage from the resin using of
cleavage cocktail (10 mL) and isolation according to the procedures
described for 5 gave 355 mg of crude material. Following reversed-
phase preparative HPLC (mobile phase gradient of 10% at 0 min–
50% at 8 min–55% at 20 min, MeCN/water containing 0.1% TFA,
tR = 14.6 min), product-containing fractions were combined and
the solution lyophilized to give 10 as a fluffy white solid. Yield
230.9 mg (0.35 mmol, 51%); HRMS (ESI-ICR) m/z calculated for
C
32H40N9O7 [M+H]+ 662.3045, observed 662.3044.
2.2.5. (S)-4-(((S)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-
3-((S)-2-((S)-2-(6-azidohexanamido)-3-(1H-indol-3-yl)
propanamido)hexanamido)-4-oxobutanoic acid (12)
A cocktail (3 mL) consisting of TFA, thioanisole, triisopropylsi-
lane, and water (9.1:0.3:0.3:0.3) was injected into the syringe reac-
tor, which was shaken for 4 h at rt. The solution was then collected
into a centrifuge tube (15 mL) and the resin washed with further
aliquots of TFA (2 ꢀ 2 mL ꢀ 2 min). The combined TFA solutions
were concentrated in the centrifuge tube under a stream of argon
and the product precipitated by the addition of cold ether (8 mL).
The tube was centrifuged and the supernatant removed. The pellet
was washed with cold ether (3 ꢀ 6 mL), air dried, dissolved in
water/MeCN, and lyophilized. The resultant solid was subjected
to reversed-phase HPLC (mobile phase gradient of 0–90% MeCN/
water containing 0.1% TFA over 45 min, tR = 22.9 min), product-
containing fractions were combined, and lyophilized to give 5 as
Using the coupling procedures described previously, compound
12 was prepared from 6-azidohexanoic acid (0.34 g, 2.04 mmol,
3 equiv) and resin-bound 4 (ꢁ0.68 mmol). Cleavage from the resin
and isolation according to the procedures described for 5 gave
421 mg of crude material. Following reversed-phase preparative
HPLC (mobile phase gradient of 10% at 0 min–50% at 10 min–90%
at 25 min, MeCN/water containing 0.1% TFA, tR = 17.7 min), pro-
duct-containing fractions were combined and the solution lyophi-
lized to provide 12 as a fluffy white solid. Yield 198 mg (0.28 mmol,
41%); HRMS (ESI-ICR) m/z calculated for C36H48N10O10 [M+H]+
718.36712, observed 718.36708.
a fluffy white solid. Yield 75.0 mg (59 lmol, 35%); HRMS (ESI-
ICR) m/z calculated for C58H84N11O21 [MꢂH]ꢂ 1270.5849, observed
2.2.6. (3S,6S,9S)-9-((1H-Indol-3-yl)methyl)-3-(((S)-1-amino-1-
oxo-3-phenylpropan-2-yl)carbamoyl)-6-butyl-5,8,11-trioxo-
13,16,19,22,25-pentaoxa-4,7,10-triazahentriacont-30-ynoic acid
(14)
1270.5832.
2.2.3. Ac-Trp-Nle-Asp-Phe-NH2 (Ac-CCK4, 6)23
To resin-bound 4 (0.34 mmol) in a polypropylene syringe
equipped with a polypropylene frit was added a solution of 20%
piperidine in DMF (8 mL) and the syringe was shaken for two
min. The solution was removed, 20% piperidine in DMF (8 mL)
was again added, and the mixture was shaken for 18 min. After
removal of the solution, the resin was washed sequentially with
DMF (3 ꢀ 8 mL), DCM (3 ꢀ 8 mL), and DMF (3 ꢀ 8 mL). A 1:1 mix-
For the synthesis of 14, procedures modified from those previous-
ly described were used to couple 3,6,9,12,15-pentaoxahenicos-20-
ynoic acid (0.70 g, 2.10 mmol, 3 equiv, synthesized as described in
Supplementary data that accompanies this article) to resin-bound
4 (ꢁ0.68 mmol). Specifically, bromophenol blue was not used, a cou-
pling time of 2 h was employed, and post-coupling acylation was not
performed. Cleavage from the resin using cleavage cocktail (10 mL)
and isolation according to the procedures described for 5 gave a solid
which was subjected to reversed-phase preparative HPLC (mobile
phase gradient of 10–90% MeCN/water containing 0.1% TFA over
45 min, tR = 16.9 min). Product-containing fractions were combined
and the solution lyophilized to produce 14 as a fluffy white solid.
Yield 243 mg (0.27 mmol, 40%); HRMS (ESI-ICR) m/z calculated for
ture of acetic anhydride (960 lL) and pyridine (820 lL) in DMF
(3 mL) was taken up into the syringe reactor, which was shaken
for 1 h. The resin was washed with DMF (3 ꢀ 8 mL), DCM
(3 ꢀ 8 mL), DMF (3 ꢀ 8 mL), THF (3 ꢀ 8 mL), and DCM (3 ꢀ 8 mL).
Cleavage from the resin and isolation according to the procedures
described for 5 gave 190 mg of crude product. Following reversed-
phase preparative HPLC (mobile phase gradient of 10% at 0 min–
50% at 8 min–55% at 20 min, MeCN/water containing 0.1% TFA,
tR = 13.3 min), product-containing fractions were combined and
the solution lyophilized to give 6 as a fluffy white solid. Yield
82 mg (0.13 mmol, 39%); mp, decomposes at 232 °C; HRMS (ESI-
ICR) m/z calculated for C32H41N6O7 [M+H]+ 621.3031, observed
621.3029; analytical HPLC tR = 17.47 min.
C
46H65N6O12 [M+H]+ 893.46550, observed 893.46609.
2.3. Synthesis of the probe Eu-DTPA-PEGO-CCK4 (3)
The metal-free precursor 5 (20.0 mg, 15.7 mol) was dissolved
l
in 0.1 M ammonium acetate solution at a concentration of 1 mg/
mL. The pH was adjusted to 7–8 with aqueous 0.1 M NH4OH and
EuCl3ꢃ6H2O (11.5 mg, 31.4
lmol, 2.0 equiv) was added. The reac-
2.2.4. (S)-4-(((S)-1-Amino-1-oxo-3-phenylpropan-2-yl)amino)-
3-((S)-2-((S)-2-(2-azidoacetamido)-3-(1H-indol-3-yl)
propanamido)hexanamido)-4-oxobutanoic acid (10)
tion mixture was stirred at rt overnight. Salts were removed using
a Sep-PakÒC18 reverse-phase (500 mg) column as previously report-
ed.13,15 Product-containing fractions were concentrated and lyo-
philized to afford the product Eu-DTPA-PEGO-CCK4 (3) as a fluffy
To a solution of sodium azide (11.5 g, 176 mmol, 2.5 equiv) in
water (60 mL) was added bromoacetic acid (10.0 g, 70.5 mmol,
1 equiv) at 0 °C and the resultant solution was stirred at rt for 24
h.24 The reaction was diluted with aqueous 1 N HCl (150 mL) and
extracted with diethyl ether (4 ꢀ 50 mL). The organic extracts were
combined, dried with anhydrous Na2SO4, filtered, and evaporated
in vacuo to yield azidoacetic acid as a clear oil. Yield 6.1 g
(60.4 mmol, 86%); Rf 0.32 (20% EtOAc/DCM, visualization
KMnO4); IR (NaCl plates, cmꢂ1) 3500–2500 (br), 2924, 2113,
white solid (16.2 mg, 11.4 lmol, 73%). The presence of unbound
europium ions was confirmed by a xylenol orange assay.25
An Empore™ chelating disk (47 mm) was placed on a sintered
glass filter holder (47 mm) base fitted to a 1 L filter flask. The sol-
vent reservoir was then clamped on to the base, completing the
apparatus. The disk was wetted with 2–5 mL of distilled water,
as per the manufacturer’s instructions. This resulted in swelling
of the disk. Nitric acid (3 M, 20 mL) was added, the disk allowed

N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
1845
to soak in this solution for 1 min, then a vacuum was applied,
drawing the acid solution through the filter. The disk was washed
on the filter under vacuum with water (2 ꢀ 50 mL). The disk was
allowed to go dry between each wash. To put the disk in its most
active ammonium form, 100 mL of 0.1 M ammonium acetate buffer
(pH 5.3, made up in HPLC grade water) was added to the solvent
reservoir and the disk allowed to soak for 1 min. The solution
was then drawn through the disk by applying a vacuum, and the
disk left to dry for 5–20 min before sample loading. During this
time, the apparatus was disassembled, cleaned thoroughly with
distilled water followed by HPLC grade water, and the apparatus
reassembled for use.
14 (80.4 mg, 90
TACP (6.7 mg, 18
19.8 mol, 1.1 equiv per TACP), and 150
The vial walls were washed down with 75
DMF, the vial sealed, and irradiated in a microwave reactor to
maintain a temperature of 100 °C for 4 h. Volatiles were removed
in vacuo and the residue dissolved in water (50 mL). The aqueous
solution was extracted with a solution of dithizone in chloroform
(0.5 mM, 3 ꢀ 30 mL) and chloroform (2 ꢀ 30 mL). The resulting
aqueous solution was lyophilized, giving 30 mg of residue.
Preparative reversed-phase HPLC (mobile phase gradient of 10%
at 0 min–60% at 13 min–70% at 20 min, MeCN/water containing
0.1% TFA, tR = 19.8 min) gave 9 as a white solid after lyophilization.
l
mol, 6 equiv), TBTA (9.5 mg, 18
l
mol, 1.2 equiv),
l
mol, 1.2 equiv), sodium ascorbate (3.9 mg,
L of argon-purged DMF.
L of argon-purged
l
l
l
A solution of 3 (15.0 mg) in water/MeCN (3 mL/mg, pH 5–7
using the minimum amount of MeCN required to fully dissolve
the compound) was passed through the disk at a flow rate of 30–
40 mL/min to allow binding interactions. The disk was then
washed with MeCN/water (50:50, 30 mL). The combined filtrates
were transferred to a round bottom flask and lyophilized to give
the purified product 3. The removal of unchelated Eu ions was con-
firmed by a xylenol orange assay.25 Recovery was 14.6 mg (97%);
Yield 7.9 mg (2.5
C
lmol, 17%); HRMS (ESI-ICR) m/z calculated for
162H234N27O39 [M+3H]3+ 1060.5714, observed 1060.5729; analyti-
cal HPLC tR = 25.12 min.
2.5. Biological assays
2.5.1. Preparation of solutions
HRMS (ESI-ICR) m/z calculated for
C
58H84EuN11O21 [M+2H]2+
Stock solutions of the CCK4 trimers 7–9, control compounds 6,
15, and 16, and the probe Eu-DTPA-PEGO-CCK4 (3) were made
up in DMSO at a nominal concentration of 2.0 mM based on mea-
sured weights of solutes. Except for the control compounds 15 and
711.7527, observed 711.7527; analytical HPLC tR = 13.82 min.
2.4. Copper-catalyzed azide–alkyne cyclization (CuAAC)
reactions
16, final concentrations were determined by comparison to a DTrp
standard solution (0.50 mM) using analytical HPLC. Selective
growth media for cell growth was prepared by supplementing
Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine
serum, 1% penicillin–streptomycin, 0.1% zeocin, and 0.8% geneticin.
Basic buffer was prepared by dissolving 5.97 g HEPES and 2 g BSA in
1 L of DMEM. The pH of this solution was adjusted to 7.4 using 2 N
2.4.1. CCK4 Trimer 7
To a microwave vial (0.5–2 mL) purged with argon were added
tripropargylamine (571
azide 10 (79.4 mg, 120
TACP (29 mg, 78 mol, 3.9 equiv), and 2,6-lutidine (470
40 mol, 2 equiv). The vial walls were washed down with 400
l
L of a 0.035 M solution in DMF, 20
l
mol),
L),
L,
l
mol, 6 equiv), argon-purged DMF (500
l
l
l
l
lL
NaOH and the solution sterilized by filtration through a 0.22 lm
of argon-purged DMF, the vial sealed, and stirred at rt for 4 days.
Volatiles were removed in vacuo and the residue dissolved in
water/MeCN (250 mL). The aqueous solution was extracted with
a solution of dithizone in chloroform (1.5 mM, 3 ꢀ 50 mL) and
chloroform (2 ꢀ 50 mL), then lyophilized. The residue was subject-
ed to preparative reversed-phase HPLC (mobile phase gradient of
10% at 0 min–55% at 15 min–80% at 25 min, MeCN/water contain-
ing 0.1% TFA, tR = 15.1 min) giving 7 as a white solid after
filter (CorningÒ, 431117, 500 mL bottle-top filter, sterile) under
vacuum. Binding buffer was prepared by supplementing DMEM
(1 L) with HEPES (5.97 g), BSA (2 g), 1,10-phenanthroline (1 mL of
a 1 M solution in EtOH), leupeptin (1 mL of a 500 mg/L aqueous
solution), and bacitracin (1 mL of a 200 g/L aqueous solution).
The pH of this solution was adjusted to 7.4 using 2 N NaOH and
the solution sterilized by filtration through a 0.22 lm filter
(Corning, 431117, 500 mL bottle-top filter, sterile) under vacuum.
lyophilization. Yield 5.2 mg (2.5 lmol, 12%); HRMS (ESI-ICR) m/z
calculated for C105H128N28O21 [M+2H]2+ 1058.48989, observed
OR
1058.49173; analytical HPLC tR = 21.18 min.
R
N
2.4.2. CCK4 Trimer 8
R
R
To a microwave vial (0.5–2 mL) purged with argon were added
RO
OR
11 (150
(80.6 mg, 112.2
1.2 equiv), TACP (8.4 mg, 22.4
(4.9 mg, 24.7 mol, 1.1 equiv per TACP), and 500
DMF. The vial walls were washed down with 400
l
L of a 0.122 M solution in DMF, 18.3
mol, 6 equiv), TBTA (11.9 mg, 22.4
mol, 1.2 equiv), sodium ascorbate
L of argon-purged
L of argon-
l
mol), azide 12
16
15
l
lmol,
l
Ser-NH2
l
l
N
N
CH2
R =
l
O
N
purged DMF, the vial sealed, and irradiated in a microwave reactor
to maintain a temperature of 100 °C for 4 h. Volatiles were removed
in vacuo and the residue dissolved in water/MeCN (100 mL). The
aqueous solution was extracted with a solution of dithizone in chlo-
roform (0.5 mM, 3 ꢀ 40 mL) and chloroform (2 ꢀ 40 mL), then lyo-
philized. The residue was subjected to preparative reversed-phase
HPLC (mobile phase gradient of 10% at 0 min–55% at 15 min–80%
at 25 min, MeCN/water containing 0.1% TFA, tR = 16.6 min) affording
2.5.2. Cell culture
HEK-293 cells expressing both hMC4R and hCCK2R18 were
maintained in selective growth media. For binding assays, cells
were plated into six-well plates (Greiner Bio-One, 657160, Cell
Culture Multi-well Plates, Polystyrene, six wells) at 240,000 cells
per well in a total volume of 3 mL (2 mL of the selective growth
media added initially to each well, followed by 1 mL of cell suspen-
sion). On the third day after plating, additional selective growth
media (1 mL) was carefully added to each well so as not to disturb
the cells, which were then left to grow until ꢁ90% confluency was
achieved (usually by Day 5) before conducting assays.
8 as a white solid after lyophilization. Yield 5.2 mg (2.2
lmol, 12%);
HRMS (ESI-ICR) m/z calculated for
C
123H155N27O24 [M+2H]2+
1197.0864, observed 1197.0872; analytical HPLC tR = 22.42 min.
2.4.3. CCK4 Trimer 9
To a microwave vial (0.2–0.5 mL) purged with argon were
added 13 (29.0 lL of a 0.518 M solution in DMF, 15 lmol), alkyne

1846
N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
2.5.3. Saturation binding assays
Following this incubation, cells and cell fragments were pellet-
L aliquots of supernatant
from each tube were transferred to a 96-well plate (Perkin–
Elmer, 6005060, tissue culture treated B&W Isoplate-96) for
fluorescence measurement using a VICTOR X4 2030 Multilabel
Reader.
Six solutions containing both the probe Eu-DTPA-PEGO-CCK4
(3) and Ac-CCK4 (6) were made in 1.2 mL of binding buffer in sepa-
rate micro-centrifuge tubes (one per each well). All six tubes con-
ed (5000 rpm for 5 min) and 4 ꢀ 100
l
tained 1 lM concentrations of 6, while the concentration of 3
varied across the six tubes (0.1, 10, 25, 50, 100, and 250 nM).
These six solutions were used to assess nonspecific binding. A sec-
ond set of six solutions (1.2 mL each) contained 3 at the same con-
centrations without the blocking ligand 6. These solutions were
used to assess total binding.
To commence an assay, cell-containing six-well plates (ꢀ2)
were removed from the incubator and the selective growth media
removed by careful aspiration. The twelve prepared solutions
detailed above were carefully transferred (1 mL per well) by pipet-
te down the well walls to minimize disturbance of the cells, which
can result in cell loss during media exchanges. The plates were
then maintained in a CO2 incubator at 37 °C for 1 h.
2.6. Data analysis
NMR data were analyzed using MestReNova software (Mestre
Lab Research S. L., version 7.1.1). Binding data analysis was per-
formed using GraphPad Prism software (version 5.04). A descrip-
tion of the binding equations used appears in Supplementary
data that accompanies this article.
3. Results
3.1. Chemistry
Solutions were then removed by careful aspiration and 600
of basic buffer were added to each well. The cells were scraped
from each well individually using Cell Scraper (18 cm,
lL
The fully protected resin-bound precursor 4 to probe 3, Ac-CCK4
(6), azides 10 and 12, and alkyne 14 was prepared by solid phase
synthesis on Rink amide AM resin (Scheme 1). For the preparation
of probe 3, Fmoc-PEGO26 was coupled to the N-terminus of the
resin-bound peptide. Following removal of the Fmoc, DTPA, acti-
vated as the HOBt ester, was attached to the N-terminus of the
PEGO spacer. Simultaneous side chain deprotection and cleavage
of the peptide from the resin followed by purification by prepara-
tive HPLC produced the metal-free precursor DTPA-PEGO-CCK4 (5)
in 35% yield. Compound 5 was characterized by ESI-ICR MS.
Following complexation of Eu3+ by 5, purification and partial
desalting by reversed-phase chromatography, final desalting by
passage through an Empore chelating disk,25 and recovery by
lyophilization afforded Eu-DTPA-PEGO-CCK4 (3) in 73% yield.
Compound 3 was characterized by analytical HPLC and ESI-ICR
a
GeneMate) and transferred in suspension to separate 1.7 mL
micro-centrifuge tubes. The wells and scraper were rinsed with
600 lL of basic buffer and the rinses combined with the corre-
sponding suspensions. One scraper was used across the wells mea-
suring total binding, and another was used across the wells
measuring non-specific binding.
The tubes containing the cell suspensions were centrifuged
(3000 rpm for 3 min) in a micro-centrifuge. After removing the
supernatant, the cells were re-suspended in basic buffer (1 mL)
and incubated for 5 min in a 37 °C water bath during each of three
wash cycles. After the final wash, 600 lL of DELFIA Enhancement
Solution (Perkin Elmer 1244-104) was added to each cell pellet,
the tubes were mixed using a vortex mixer, and incubated for
1 h in a water bath maintained at 37 °C.
MS. The absence of unchelated Eu3+ ion (within a 10
lM limit of
Following this incubation, cells and cell fragments were pellet-
detection) was confirmed by means of a xylenol orange spec-
ed (5000 rpm for 5 min) and 4 ꢀ 100
lL aliquots of supernatant
trophotometric assay.25
from each tube were transferred to a 96-well plate (Perkin–
Elmer, 6005060, tissue culture treated B&W Isoplate-96) for
fluorescence measurement using a VICTOR X4 2030 Multilabel
Reader.
For preparation of 6, the resin-bound tetrapeptide 4 was N-ter-
minally acetylated. Side chain deprotection and cleavage of the
peptide from the resin, purification by reversed-phase preparative
HPLC, and recovery by lyophilization gave
6 in 39% yield.
Compound 6 was characterized by analytical HPLC and ESI-ICR MS.
Compounds 7 and 8 were prepared as depicted in Scheme 2.
Tripropargylamine is commercially available. The synthesis of
phloroglucinol derivative 11 was previously described.21 Azides
10 and 12 were prepared by coupling 2-azidoacetic acid and 6-azi-
dohexanoic acid,27 respectively, to the N-terminus of the resin-
bound peptide 4 (chemistry not depicted). Side chain deprotection
and cleavage of the peptides from the resin, purification by
reversed-phase preparative HPLC, and recovery by lyophilization
produced 10 and 12 in 51% and 41% yields, respectively. These
compounds were characterized by ESI-ICR MS. The copper-cat-
alyzed azide–alkyne cyclization (CuAAC) reaction of tripropargy-
lamine with 10 at room temperature produced 7. The CuAAC
reaction of 11 with 12 under microwave irradiation to maintain
a temperature of 100 °C produced 8. In each case, copper ions were
removed from the crude product mixture by complexation with
dithizone with removal of the resulting complex by extraction with
CHCl3.28 The water-soluble material was then recovered by
lyophilization, subjected to preparative reversed-phase HPLC, and
product-containing fractions subjected to lyophilization to give
products 7 and 8, each in 12% yield.
2.5.4. Competitive binding assays
Immediately before an assay, twelve solutions (1350 lL each) of
compounds to be tested were made up in binding buffer in micro-
centrifuge tubes at concentrations ranging from 10ꢂ5 to 10ꢂ12 M.
Each of the tubes also contained the probe Eu-DTPA-PEGO-CCK4
(3) at a concentration of 15 nM. Cell-containing six-well plates
(ꢀ2) were removed from the incubator and the selective growth
media removed by careful aspiration. The twelve prepared solu-
tions from above were carefully transferred (1 mL per well) by
pipette down the well walls. The plates were then maintained in
a CO2 incubator at 37 °C for 1 h.
After 1 h, solutions were removed by careful aspiration and
600 lL of basic buffer were added to each well. The cells were
scraped from each well using a Cell Scraper (18 cm, GeneMate)
and transferred in suspension to separate 1.7 mL micro-centrifuge
tubes. The wells and scraper were rinsed with 600 lL of basic buf-
fer and the rinses combined with the corresponding suspensions.
The cell suspensions were centrifuged (3000 rpm for 3 min) in a
micro-centrifuge. After removing the supernatant, the cells were
re-suspended in basic buffer (1 mL) and incubated for 5 min in a
37 °C water bath during each of three wash cycles. After the final
Compound
9 was prepared as depicted in Scheme 3.
wash, 600 lL of DELFIA Enhancement Solution (Perkin Elmer
Phloroglucinol derivative 13 was prepared as described in
Supplementary data that accompanies this article. Alkyne 14 was
prepared by coupling 3,6,9,12,15-pentaoxahenicos-20-ynoic acid
1244-104) was added to each cell pellet, the tubes were mixed
using a vortex mixer, and incubated in a water bath maintained
at 37 °C for 1 h.

N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
1847
(prepared as described in Supplementary data) to the N-terminus
of the resin-bound peptide 4. Side chain deprotection and cleavage
of the peptide from the resin, purification by reversed-phase pre-
parative HPLC, and recovery by lyophilization produced 14 in
40% yield. This compound was characterized by ESI-ICR MS. The
CuAAC reaction of 13 with 14 under microwave irradiation to
maintain a temperature of 100 °C produced 9. Copper ions were
removed from the crude product mixture by complexation with
dithizone with removal of the resulting complex by extraction with
CHCl3.28 The water-soluble material was then recovered by
lyophilization, subjected to preparative reversed-phase HPLC, and
product-containing fractions subjected to lyophilization to give 9
in 17% yield.
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
6
7
8
9
-12
-10
-8
-6
-4
[log] Concentration
Compounds 7-9 were characterized by analytical HPLC and ESI-
ICR MS.
Figure 2. Competitive binding curves for the compounds 6–9 against probe 3
(15 nM). Control compounds 15 and 16 were not competitive inhibitors of 3 over
the concentration range tested.
3.2. Bioassays
HEK-293 cells overexpressing both hCCK2R29 and hMC4R30,31
were used to measure the affinity of probe 3 for binding to
hCCK2R by means of saturation binding assays (Fig. 1).21 Ac-
CCK4 (6)23 was used as the blocking ligand. The Kd value obtained
for probe 3 was 17 2 nM.
Competitive binding assays were conducted for compounds 6–
9, 15, and 16 against probe 3. Sample binding curves appear in
Figure 2 and Ki values are listed in Table 1.
Table 1
Results of competitive binding assaysa
Compound
Ki SEMb (nM)
Relative binding affinityc
Ac-CCK4 (6)
7
8
9
15
16
6.1 2.1
1.2 0.4
3.7 0.4
1
5
2
0.2
nae
nae
30
8
nbd
nbd
a
4. Discussion
Competition experiments were carried out against probe
3 (Kd = 17 nM,
[3] = 15 nM) using HEK-293 cells overexpressing hCCK2R and hMC4R.
b
SEM = standard error of the mean; n = 4 independent determinations.
Ratio of the Ki of Ac-CCK4 (6) to the compound Ki value.
nb = no competitive binding observed.
A recent study of TRF probes for melanocortin receptors showed
that probes based on ligands with less than the maximal binding
affinity offered greater ease of synthesis and, in some cases, excel-
lent performance in competitive binding assays.15 With this in
mind, development and testing of a probe for cholecystokinin
c
d
e
na = not applicable.
receptors based on the minimal active sequence Trp-Met-Asp-
32,33
purification by reversed-phase HPLC afforded 5 in 35% yield.
Treatment of 5 with excess EuCl3 in a pH 7–8 buffer gave the che-
late 3, but passage through a Sep-PakÒC18 column left unchelated
europium ions as a contaminant as determined by a xylenol orange
assay.25 Complete removal of unchelated europium ions is essen-
tial to obtaining reliable bioassay readings. Fortunately, passage
of 3 through an Empore chelating disk removed the unchelated
europium ions.25 The overall yield of 3 was 26%, significantly
higher than the 16% yield reported for the synthesis of probe 1.16
Saturation binding assays were conducted to characterize the
affinity of probe 3 for CCK2R using HEK-293 cells that overexpress
both hCCK2R and hMC4R. Despite having a highly truncated ligand
relative to probe 1, probe 3 bound tightly to CCK2R. The Kd for
tetrapeptide 3 was 17 nM, while the reported Kd for octapeptide
1 is 35 nM.18
Phe-NH2
was undertaken.
Eu-DTPA-PEGO-CCK4 (3) was synthesized via a solid phase pep-
tide synthesis strategy (Scheme 1). The protected tetrapeptide 4
was assembled on Rink amide AM resin. Methionine was replaced
with the isosteric analog norleucine, which is not susceptible to
oxidation and is less prone to proteolytic degradation.34,35 This
substitution was expected to afford a probe with increased shelf
life34,36 and high biological activity.36–38 A PEGO spacer was used
to minimize interference of the europium chelate with binding of
the ligand to the receptor. Given the hydrophobic nature of
CCK4, this was expected to enhance the water solubility of the
probe. Following deprotection and cleavage from the resin,
Probe 3 was developed for use in high-throughput screening of
unlabeled molecules for binding to CCK receptors. To establish its
efficacy for this purpose, trivalent compounds 7–9 were synthe-
sized and tested against 3 in competitive binding assays. Three
quite different inter-ligand distances were employed in 7–9, since
the optimum ligand spacing for multivalent binding to CCK recep-
tors has not been established. All three compounds were prepared
by a combination of solid phase synthesis (from 4) and copper-cat-
alyzed azide–alkyne cyclization as depicted in Schemes 2 and 3.
Positive control 6 and negative controls 1521 and 1621 in which
serinamide residues replace the CCK4 ligands were also competed
against probe 3. Results of the competitive binding assays are given
in Table 1.
5
4
3
2
1
0
0
100
200
300
Concentration (nM)
As was expected, compounds 15 and 16 did not interfere with
the binding and uptake of probe 3. Control 6 exhibited a Ki of
Figure 1. Saturation binding curves for probe 3. Total binding ( ), non-specific
binding ( ), and specific binding ( ). The calculated Kd = 17 2 nM (n = 5).
6.1 nM,
a value consistent with those reported for other

1848
N. G. R. Dayan Elshan et al. / Bioorg. Med. Chem. 23 (2015) 1841–1848
monovalent CCK4 ligands.20,39 Given the high affinity of CCK4,
avidities indicative of multivalent binding were not expected for
CCK4 trimers 7–9. Even if the ligand spacing had been optimal,
enhanced avidities due to multivalent binding are demonstrable
only when weakly binding ligands are employed.4,5,20 Modest
increases in binding affinity were observed for compounds 7 and
8 relative to 6 that can be attributed to statistical and/or proximity
effects. Compound 9 exhibited a significantly lower binding affinity
compared to CCK4 trimers 7 and 8. This maybe due to a larger
entropy penalty for 9 upon binding to CCK2R or to hydrophobic
interactions between the PEG spacer and the CCK4 ligand which
hinder binding to the receptor.40,41 For these reasons, lower affinity
ligands are currently being identified for incorporation into multi-
valent molecules targeting CCK2R.
5. Kiessling, L. L.; Lamanna, A. C. NATO Sci. Ser., II: Math. Phys. Chem. 2003, 129,
345.
6. Behr, T. M.; Jenner, N.; Béhé, M.; Angerstein, C.; Gratz, S.; Raue, F.; Becker, W. J.
Nucl. Med. 1999, 40, 1029.
7. Miller, L. J.; Rosenzweig, S. A.; Jamieson, J. D. J. Biol. Chem. 1981, 256, 12417.
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14. Xu, L.; Vagner, J.; Alleti, R.; Rao, V.; Jagadish, B.; Morse, D. L.; Hruby, V. J.;
Gillies, R. J.; Mash, E. A. Bioorg. Med. Chem. Lett. 2010, 20, 2489.
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Tafreshi, N. K.; Brabez, N.; Weber, C. S.; Lynch, R. M.; Hruby, V. J.; Gillies, R. J.;
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5. Conclusion
17. Handl, H. L.; Gillies, R. J. Life Sci. 2005, 77, 361.
A short and efficient synthesis of a new TRF probe that binds to
cholecystokinin-2 receptors has been developed. The combination
of this probe with the high expression levels of CCK2R (ꢁ1.1 ꢀ 106
receptors/cell) by the engineered HEK-293 cell line and the use of
large cell populations (ꢁ1 ꢀ 106 cells per well at 90% confluence) in
the six-well plate assay employed provide binding assay data with
very high signal-to-noise ratios. Hence, the Eu-DTPA-PEGO-CCK4
probe 3 could become an important tool for high-throughput
screening of binding to cholecystokinin receptors.
18. Xu, L.; Vagner, J.; Josan, J.; Lynch, R. M.; Morse, D. L.; Baggett, B.; Han, H.; Mash,
E. A.; Hruby, V. J.; Gillies, R. J. Mol. Cancer Ther. 2009, 8, 2356.
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Chassaing, G.; Lavielle, S.; Hruby, V. J. Bioorg. Med. Chem. Lett. 2013, 23, 2422.
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Acknowledgements
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(last
accessed
The authors thank Renata Patek and Professor Josef Vagner for
assistance and useful discussions. The HEK-293 cell line overex-
pressing hCCK2R and hMC4R was the generous gift of Professor
Robert J. Gillies, Professor David L. Morse, and Dr. Liping Xu of
the H. Lee Moffitt Cancer Center and Research Institute, Tampa,
FL, USA. This work was supported by grants R33 CA95944, RO1
CA97360, RO1 CA123547, and P30 CA23074 from the National
Cancer Institute.
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35. Meade, A. J.; Meloni, B. P.; Mastaglia, F. L.; Knuckey, N. W. J. Exp. Stroke Transl.
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Supplementary data, including details of the synthesis of 13 and
of 3,6,9,12,15-pentaoxahenicos-20-ynoic acid (used in the synthe-
sis of 14), 1H and 13C NMR spectra of new compounds not contain-
ing CCK4, copies of the HPLC chromatograms for compounds 3 and
6–9, and details of the binding assays with binding curves associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2015.02.028.
36. Laverman, P.; Sosabowski, J. K.; Boerman, O. C.; Oyen, W. J. G. Eur. J. Nucl. Med.
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References and notes
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