Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors

Article abstract of DOI:10.1016/j.bmc.2013.06.052

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining K_d values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K _d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The K_i values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the K_i values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

Full text of DOI:10.1016/j.bmc.2013.06.052

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and Characterization of Time-resolved Fluorescence Probes
for Evaluation of Competitive Binding to Melanocortin Receptors
Ramesh Alleti a, Josef Vagner b, Dilani Chathurika Dehigaspitiya a, Valerie E. Moberg c, N. G. R. D. Elshan a,
Narges K. Tafreshi c, Nabila Brabez a, Craig S. Weber d, Ronald M. Lynch b,d, Victor J. Hruby a,b
,
Robert J. Gillies c, David L. Morse c, Eugene A. Mash a,
⇑
a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0041, USA
b The Bio5 Institute, University of Arizona, Tucson, AZ 85721-0240, USA
c Department of Functional and Molecular Imaging, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA
d Department of Physiology, University of Arizona, Tucson, AZ 85724-5051, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based
on the parental ligands MSH(4), MSH(7), and NDP- -MSH were prepared by solid phase synthesis meth-
Received 30 April 2013
Revised 14 June 2013
Accepted 22 June 2013
Available online xxxx
a
ods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells
engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecys-
tokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high
concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was
discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-
DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low
nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-
Keywords:
Competition binding assays
Fluorescent probes
Melanocortin 4 receptor
Saturation binding assays
Time-resolved fluorescence
DTPA-PEGO probes based on MSH(7) and NDP-a-MSH exhibited Kd values of 27 3.9 nM and
4.2 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding
assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and
NDP-a-MSH constructs derived from squalene. Results from assays with both probes reflected only sta-
tistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent con-
structs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were
generally lower than the Ki values obtained when the probe based on NDP-a-MSH was employed, which
is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed
with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent
binding in competitive binding assays against a variant of the probe based on NDP-a-MSH. Results from
these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4)
constructs than was previously reported.
Ó 2013 Published by Elsevier Ltd.
1. Introduction
Abbreviations: BSA, bovine serum albumin; Cl-HOBt, 6-chloro-1-hydroxyben-
zotriazole; CuAAC, copper(I)-catalyzed azide-alkyne cycloaddition; DCM, dichloro-
methane; DIC, diisopropyl carbodiimide; DIEA, diisopropylethylamine; DMEM,
Dulbecco’s Modified Eagle Medium; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; IC50, half maximal
inhibitory concentration; ESI MS, electrospray ionization mass spectrometry;
Fmoc, 9-fluorenylmethyoxycarbonyl; Fmoc-PEGO, 1-(9H-fluoren-9-yl)-3,19-dioxo-
2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic acid; FT-ICR MS, Fourier trans-
form ion cyclotron resonance mass spectrometry; hMC4R, human melanocortin 4
receptor; HOBt, 1-hydroxybenzotriazole; HRMS, high resolution mass spectros-
copy; MEM, Minimum Essential Medium; MSH(4), His-DPhe-Arg-Trp; MSH(7), Ser-
The affinity of a molecule for binding to a receptor is often
quantified by a competitive binding assay against a labeled ligand
of known potency. For example, labeled forms of Ac-Ser-Tyr-Ser-
Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 [NDP-a-MSH], a
superpotent ligand that binds to melanocortin receptors,1,2 have
been used for this purpose.3,4 In such assays, one often assumes
thermodynamic control, i.e., that the respective on-rates and off-
rates of both the competing and competed ligands are similar so
that all bound and unbound states are in equilibrium. If this is
not the case, details of how the assay is carried out (order and tim-
ing of reagent addition, timing of measurements taken) can affect
the outcome. Determination of on-rates and off-rates for binding
of molecules to living cells is difficult since ligands and labeled
Nle-Glu-His-DPhe-Arg-Trp; NDP-a-MSH, Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-
Trp-Gly-Lys-Pro-Val-NH2; PEGO, 19-amino-5-oxo-3,10,13,16-tetraoxa-6-azanon-
adecan-1-oic acid; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine;
TEAA, triethylammonium acetate; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TLC,
thin-layer
chromatography;
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).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.06.052
Please cite this article in press as: Alleti, R.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.052

2
R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
probes may be taken up by the cells and because receptors may
cycle to and from the cell surface. In the absence of knowledge of
ligand and probe on-rates and off-rates, a close match between
the affinities of the competed probe and the competing ligand
in a competitive binding assay would seem prudent—but is it nec-
essary? This issue was examined experimentally as reported
herein.
competitive binding assays.14 The Kd of 2, determined by
saturation binding to HEK-293 cells overexpressing hMC4R, was
9.1
herein syntheses of the structurally related probes Eu-DTPA-PEGO-
MSH(7) and Eu-DTPA-PEGO-NDP- -MSH 4, studies of the
l
M, compared with a reported Kd for 1 of 8.3 nM.15 We report
3
a
saturation binding of 2–4 with hMC4R, and the use of 3 and 4 in
competitive binding assays involving monovalent and divalent
We have investigated the binding of multivalent molecules,
including several derived from the weak ligand Ac-His-DPhe-Arg-
MSH(4), MSH(7), and NDP-a-MSH constructs derived from
squalene16, as well as monovalent, divalent, and trivalent MSH(4)
constructs that previously exhibited multivalent binding in
competitive binding assays against a variant of probe 4.17
Trp-NH2 [MSH(4)]5–7
,
to human melanocortin
4 receptors
(hMC4R).8 MSH(4) was selected because synergistic effects are
generally more easily detected for multivalent constructs of
low-affinity ligands.9–13 Probe 1 is based on NDP-
-MSH and was
2. Materials and methods
2.1. Chemical synthesis
a
used to determine the Ki values for many of our multivalent
constructs. However, we became concerned that competitions
between superpotent probes such as 1 and multivalent constructs
based on much weaker ligands such as MSH(4) were inherently
unbalanced, and that perhaps the measured avidity of a competing
multivalent construct depended on the affinity of the competed
2.1.1. General experimental
Dichloromethane (DCM), diethyl ether, and tetrahydrofuran
(THF) were dried by passage through activated alumina. Other sol-
vents and commercial reagents were used as supplied. For mois-
ture sensitive reactions, glassware was flame-dried under argon.
Analytical thin-layer chromatography (TLC) was carried out on
fluorescent probe. In
a preliminary study, we prepared the
Eu-DTPA-PEGO-MSH(4) probe 2 and tested it in saturation and
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R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
pre-coated silica gel 60 F-254 plates with visualization by UV expo-
sure, by exposure to I2 vapor, or by staining with 10% phosphomo-
lybdic acid solution in ethanol or with 5% H2SO4 in ethanol and
heat. Gravity column chromatography was accomplished using sil-
groups was confirmed by the Kaiser test. The above cycle of proce-
dures was repeated for coupling of the other amino acids in the se-
quence, producing the resin-bound peptide derivatives. Further
details for production of probes 2–4 and azides 5–7 are given in
sections 2.1.2.1. and 2.1.2.2.
ica gel 60 (63–210 lm). NMR spectra were recorded at 300 MHz or
500 MHz for 1H NMR and at 75 MHz or 125 MHz for 13C NMR.
Chemical shifts (d) are expressed in ppm and are internally refer-
enced (7.24 ppm for CDCl3 and 3.31 ppm for CD3OD for 1H NMR
and 77.0 ppm for CDCl3 and 49.15 ppm for CD3OD for 13C NMR).
2.1.2.1. Synthesis and characterization of probes 2–
4.
Attachment of the PEGO linker was performed using DIC/
HOBt activation (3 equiv Fmoc-PEGO18, 3 equiv of HOBt, and
3 equiv of DIC). Next, the DTPA chelator was attached to the N-ter-
minus of the resin–bound construct as follows. After Fmoc re-
moval, the resin was washed with DMSO. DTPA dianhydride19
(10 equiv) and HOBt (30 equiv) were dissolved in dry DMSO
(1 mL) at 50 °C and then stirred for 20 min at rt. This mixture
was injected into the syringe reactor which was shaken overnight,
then the resin washed with DMSO, THF, 20% aqueous THF, THF, 5%
DIEA in THF (5 min), THF, DMF, THF, and DCM. A cleavage mixture
consisting of trifluoroacetic acid, water, 1,2-ethanedithiol, and
thioanisole (91:3:3:3, 10 mL/g of resin) was injected into the syr-
inge reactor containing the resin and the mixture shaken for 4 h
at rt. The solution was then filtered off and the resin washed twice
with TFA. Filtrates were collected, concentrated under a stream of
nitrogen, and the product was precipitated by addition of cold
ether to the residue. The peptide pellet was washed three times
with cold ether, dried, dissolved in 1.0 M acetic acid, and lyophi-
lized. The lyophilized DTPA-PEGO-MSH(4), DTPA-PEGO-MSH(7),
Preparative HPLC was performed on
a
19 Â 256 mm Waters
X-Bridge Preparative C18 column. The mobile phase was 10–90%
acetonitrile and water containing 0.1% trifluoroacetic acid (TFA)
within 50 min. The flow rate was 15 mL/min. The dual UV detector
system operated at 230 and 280 nm. ESI experiments were per-
formed on an ESI Bruker Apex Qh 9.4 T FT-ICR instrument using
standard ESI conditions. The samples were dissolved in acetoni-
trile/water 1:1 containing 0.1% formic acid in a concentration
range of 1–30 lM.
2.1.2. Solid phase synthesis
For the synthesis of probes 2–4, resin-bound MSH(4), MSH(7),
a
and NDP-
a
-MSH were synthesized manually using an N -Fmoc/t-
Bu solid-phase peptide synthesis strategy and standard DIC/HOBt
activation on Rink amide Tentagel resin (approximately
0.24 mmol of active sites per gram). For the synthesis of ligands
5–7, 16, and 17, resin-bound MSH(4), MSH(7), and NDP- -MSH
S
a
a
were synthesized manually using an N -Fmoc/t-Bu solid-phase
peptide synthesis strategy and standard DIC/Cl-HOBt activation
on Rink amide resin (approximately 0.68 mmol of active sites per
gram) as follows. Resin (1.0 g) in a syringe (polypropylene reaction
tube equipped with a polypropylene frit) was allowed to swell in
THF for 1 h. THF was removed, and 20% piperidine in DMF
(15 mL) was added to deprotect the Fmoc functionality. After
2 min, the DMF/piperidine solution was removed, 20% piperidine
in DMF (15 mL) was again added, and the mixture was shaken
for 18 min. The DMF/piperidine solution was removed and the re-
sin washed with DMF (3 Â 15 mL), DCM (3 Â 15 mL), DMF
(3 Â 15 mL), 0.5 M HOBt in DMF (1 Â 15 mL), 0.5 M HOBt in DMF
(1 Â 15 mL) plus a drop of 0.01 M bromophenol blue in DMF,
DMF (2 Â 15 mL), and DCM (1 Â 15 mL), in that order. For Rink
amide resin, a mixture of the appropriate Fmoc-amino acid
(3 equivalents), Cl-HOBt (3 equivalents), and DIC (6 equivalents)
in DMF (15 mL) was allowed to react for 2 min, was then added
to the resin, and the mixture shaken for 1 h, during which time
the blue color disappeared. The resin was then washed with DMF
(3 Â 15 mL), DCM (3 Â 15 mL), and DMF (3 Â 15 mL). Free NH2
groups were capped by addition of a 1:1 mixture of acetic anhy-
dride and pyridine (6 mL). After the mixture was shaken for
20 min, the resin was washed with DMF (3 Â 15 mL), DCM
(3 Â 15 mL), and DMF (3 Â 15 mL). The absence of free amine
and DTPA-PEGO-NDP-a-MSH constructs were purified by prepara-
tive HPLC and characterized by FT-ICR MS.
The metal-free precursors were dissolved in 0.1 M ammonium
acetate, the pH was adjusted to 8 with aqueous 0.1 M NH4OH,
and 3 equiv of EuCl3ꢀ6H2O in water were added. The reaction mix-
ture was stirred at rt overnight. The excess EuCl3 and ammonium
salts were removed using a Sep-PakÒ C18 reverse-phase column
with repetitive washing (20 mL of HPLC grade water). The final
products 2–4 were eluted using 50% aqueous acetonitrile (4 mL),
concentrated, lyophilized, and characterized by analytical HPLC
and FT-ICR MS. Data appear in Table 1.
2.1.2.2. Synthesis and characterization of azides 5–7.
Azide
5 was prepared from the corresponding resin-bound tetrapeptide
and 6-azidohexanoic acid as previously described.20 Azides 6 and
7 were prepared in a manner similar to 5. Cleavage from the resin
and deprotection were achieved using a cleavage mixture of triflu-
oroacetic acid, triisopropylsilane, thioanisole, and water (91:3:3:3).
The mixture of cleavage cocktail and resin was shaken overnight,
the solution was separated from the resin, volatiles were evapo-
rated, the residue triturated with ether, and the crude product sep-
arated by centrifugation. Following purification by preparative
HPLC, compounds 5–7 were characterized by ESI MS. Data appear
in Table 1.
Table 1
MS and HPLC characterization of compounds 2–7.
Compound
Formula [M]
Calculated Masses [Ion]
Masses Found (error)
tR (min)
Yields (%)
20
2a
C60H85N16O19151Eu
1486.5532 [M + 1]+
1488.5546 [M + 1]+
1815.7119 [M + 1]+
1817.7133 [M + 1]+
817.0254 [M + 3]3+
817.6925 [M+3]3+
783.4161 [M+1]+
556.7910 [M+2]2+
872.4575 [M+2]2+
1486.5526 (0.4 ppm)
1488.5549 (0.2 ppm)
1815.7113 (0.3 ppm)
1817.7139 (0.3 ppm)
817.0248 (0.7 ppm)
817.6926 (0.1 ppm)
783.4158 (0.4 ppm)
556.7909 (0.3 ppm)
872.4567 (0.9 ppm)
10.5
C
60H85N16O19153Eu
3a
4a
C74H108N19O25151Eu
74H108N19O25153Eu
C104H155N26O33151Eu
104H155N26O33153Eu
12.9
12.6
9
C
11
C
5b
6b
7b
C38H50N14O5
C52H73N17O11
C82H118N24O19
13.6
15.4
14.6
39–45
42–50
50–60
a
Analyzed on a 3 Â 150 mm 3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear gradient from 10–60% B in A over 45 min, where A is 0.1% TEAA in water (pH 6)
and B is 90% acetonitrile and 10% A, detection at 220 and 280 nm. Purity > 95%. Characterized by FT-ICR MS.
b
Analyzed on a 3 Â 150 mm 3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in water and B is
0.1% TFA in acetonitrile, detection at 220 and 280 nm. Purity > 95%. Characterized by ESI MS.
Please cite this article in press as: Alleti, R.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.052

4
R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
data for 9: IR (neat) cmÀ1 3350 (br), 2955, 2117, 1462, 1378; 1H
NMR (500 MHz, CDCl3) 0.80–1.00 (approximately 24H, overlapping
methyl doublets), 1.10–1.22 (approximately 6H, methyl singlets),
1.10–1.80 (approximately 29H, m), 1.96 (1H, t, J = 2.5 Hz), 2.22
(2H, td, J = 7.0 Hz, 2.5 Hz), 3.30–3.50 (approximately 8H, m); HRMS
(ESI) calcd for C36H71O6 (M + H)+ 599.5245, found 599.5241. Spec-
tral data for 10: IR (neat) cmÀ1 3350 (br), 2955, 2117, 1462, 1376;
1H NMR (500 MHz, CDCl3) 0.80–1.00 (approximately 24H,
overlapping methyl doublets), 1.10–1.22 (approximately 6H,
methyl singlets), 1.10–1.90 (approximately 32H, m), 1.92–1.95
(2H, m), 2.16–2.22 (4H, m), 2.90–3.60 (approximately 10H, m);
HRMS (ESI) calcd for C42H79O6 (M + H)+ 679.5871, found 679.5862.
2.1.4. Multimer synthesis and characterization (Scheme 1)
2.1.4.1. Synthesis and characterization of 12a.
of alkyne 9 (30 mg, 50 mol), TBTA (3 mg, 6
To a mixture
mol), and tetra-
l
l
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
dry methanol (1 mL) was added serinamide azide 1120 (18 mg,
75 mol). The reaction mixture was subjected to microwave irradi-
lmol) in
2.1.3. Scaffold synthesis and characterization (Scheme 1)
2.1.3.1. Synthesis and characterization of 2,6,10,15,19,23-hex-
l
ation (Biotage Initiator, 100 °C) and the reaction monitored by TLC
(20% MeOH/CHCl3). After 4 h, volatiles were evaporated and the
residue loaded onto a silica gel 60 column. Elution with 5%
amethyltetracosane-3,7,11,14,18,22-hexaol (8).
In a three-
necked flask a solution of borane-tetrahydrofuran complex in
THF (1 M, 132 mL, 132 mmol) was deoxygenated with argon gas.
The flask was immersed in an ice bath, and 2-methyl-2-butene
(2 M, 132 mL, 264 mmol) also deoxygenated with argon gas was
added to the borane solution dropwise with stirring at 0 °C. The
resulting disiamyl borane was maintained at 0 °C for 3 h prior to
use. A solution of squalene (3.0 gm, 7.3 mmol) in THF was then
added dropwise at 0 °C. The mixture was stirred at 0 °C for 5 h,
kept at 4 °C for 3 d, then 3 N NaOH (80 mL) and 30% H2O2
(80 mL) were added. The reaction mixture was stirred at rt for
12 h and was then diluted with EtOAc (500 mL), washed with
water (2 Â 200 mL), brine (2 Â 100 mL), dried (Na2SO4), filtered,
and concentrated in vacuo to leave a viscous oil. The oil was sub-
jected to gravity column chromatography on silica gel 60 eluted
with chloroform–methanol (95:5), giving 1.94 g (51%) of 8 as a
white, foamy solid, mp 42–46 °C, Rf 0.3 (silica gel 60, 1:9
MeOH:CHCl3). IR (neat) cmÀ1 3350 (br), 2956, 1464, 1376. NMR
spectra are complex since the product is a mixture of regioisomers
and stereoisomers. The 1H NMR and 13C NMR spectra appear in the
Supplementary Data that accompanies this paper. Significant lines
from the 1H NMR spectrum are listed here. 1H NMR (500 MHz,
CDCl3) 0.85–0.97 (approximately 24H, overlapping methyl dou-
blets), 1.10–1.21 (approximately 6H, methyl singlets), 1.20–1.80
(approximately 26H, m), 3.30–3.50 (approximately 6H, m); HRMS
(ESI) calcd for C30H63O6 (M + H)+ 519.4619, found 519.4619.
MeOH/CHCl3 afforded 22 mg (26 lmol, 52%) of 12a as an gummy
solid, Rf 0.3 (silica gel 60, 1:9 MeOH/CHCl3). IR (neat) cmÀ1 3350
(br), 2933, 1677, 1546, 1461, 1377. NMR spectra are complex since
the product is a mixture of regioisomers and stereoisomers. The 1H
NMR and 13C NMR spectra appear in the Supplementary Data that
accompanies this paper. Significant lines from the 1H and 13C NMR
spectra are listed here. 1H NMR (500 MHz, CD3OD) 0.85–0.94
(approximately 24H, overlapping methyl doublets), 1.12–1.19
(approximately 6H, methyl singlets), 1.20–1.80 (approximately
29H, m), 1.92 (2H, pentet, J = 7 Hz), 2.29 (2H, t, J = 7 Hz), 2.72
(2H, t, J = 7 Hz), 2.90–3.50 (approximately 7H, m), 3.73–3.81 (2H,
m), 4.36 (2H, t, J = 7 Hz), 4.39–4.44 (1H, m), 7.74 (1H, s); 13C
NMR (125 MHz, CD3OD) 15.9, 17.8, 18.1, 19.5, 19.6, 19.7, 26.1,
26.2, 26.3, 27.1, 27.5, 27.6, 29.4, 29.7, 30.9, 31.1, 32.8, 34.9, 36.6,
36.8, 39.4, 40.3, 51.2, 52.4, 56.5, 63.2, 73.4, 77.7, 78.0, 78.3, 79.1,
79.4, 79.6, 123.3, 175.2. 176.0; HRMS (ESI) calcd for C45H88N5O9
(M + H)+ 842.6577, obsd 842.6582.
2.1.4.2. Synthesis and characterization of 12b.
of alkyne 10 (25 mg, 36 mol), TBTA (3 mg, 6
To a mixture
mol), and tetra-
l
l
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
dry methanol (1 mL) was added serinamide azide 1120 (22 mg,
90 mol). The reaction mixture was subjected to microwave
lmol) in
l
irradiation (Biotage Initiator, 100 °C) and the reaction monitored
by TLC (20% MeOH/CHCl3). After 4 h, volatiles were evaporated
and the residue loaded onto a silica gel 60 column. Elution with
2.1.3.2. Synthesis and characterization of alkynes
10. To a suspension of NaH (113 mg, 4.74 mmol) in DMF
9 and
(10 mL) was added a solution of 8 (821 mg, 1.58 mmol) in DMF
(5 mL) and the mixture was stirred at rt for 15 min. Tetrabutylam-
monium iodide (294 mg, 0.8 mmol) and a solution of 1-bromo-5-
hexyne21 (1.27 g, 7.90 mmol) in DMF (3 mL) were added to the
reaction mixture and stirring was continued for 24 h. The mixture
was then diluted with ether (150 mL), washed with water
(3 Â 50 mL), brine (20 mL), dried (Na2SO4), filtered, and concen-
trated in vacuo to give a pale yellow oil. The oil was subjected to
gravity column chromatography on silica gel 60 eluted with 2%
MeOH/CHCl3, giving 220 mg (21%) of bisalkyne 10 as a viscous
gum, Rf 0.7 (silica gel 60, 1:9 MeOH/CHCl3). Further elution of
the column with 5% MeOH/CHCl3 gave 410 mg (43%) of mono-
alkyne 9, also as a viscous gum, Rf 0.4 (silica gel 60, 1:9 MeOH/
CHCl3). NMR spectra are complex since the products are mixtures
of regioisomers and stereoisomers. The 1H NMR and 13C NMR spec-
tra appear in the Supplementary Data that accompanies this paper.
Significant lines from the 1H NMR spectra are listed here. Spectral
10% MeOH/CHCl3 afforded 38 mg (32 lmol, 90%) of 12b as a
gummy solid, Rf 0.5 (silica gel 60, 2:8 MeOH/CHCl3). IR (neat)
cmÀ1 3335 (br), 2936, 1658, 1546, 1461, 1374. NMR spectra are
complex since the product is a mixture of regioisomers and
stereoisomers. The 1H NMR and 13C NMR spectra appear in the
Supplementary Data that accompanies this paper. Significant lines
from the 1H and 13C NMR spectra are listed here. 1H NMR
(500 MHz, CD3OD) 0.85–0.94 (approximately 24H, overlapping
methyl doublets), 1.12–1.19 (approximately 6H, methyl singlets),
1.20–1.80 (approximately 35H, m), 1.91 (4H, pentet, J = 7 Hz),
2.29 (4H, t, J = 7 Hz), 2.72 (4H, t, J = 7 Hz), 2.80–3.50
(approximately 10H, m), 3.73–3.80 (4H, m), 4.36 (4H, t, J = 7 Hz),
4.41 (2H, t J = 5 Hz), 7.75 (2H, s); 13C NMR (125 MHz, CD3OD)
16.0, 18.1, 18.8, 19.0, 19.6, 26.1, 26.3, 27.2, 27.6, 30.9, 31.1, 36.6,
51.2, 56.6, 63.3, 73.4, 77.7, 123.3, 175.2, 176.0; HRMS
(ESI) calcd for
583.4305.
C
60H114N10O12 (M + 2H)2+ 583.4303, obsd
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R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
2.1.4.3. Synthesis and characterization of 13a.
of alkyne 9 (20 mg, 33 mol), TBTA (3 mg, 6
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
To a mixture
mol), and tetra-
with water (25 mL) and extracted (3 Â 30 mL) with a solution of
dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was then
washed with CHCl3 (2 Â 20 mL) and lyophilized to give a white so-
lid. Purification by HPLC (stationary phase 19 Â 256 mm X-Bridge
Preparative C18 column, mobile phase 10–90% acetonitrile and
water containing 0.1% TFA within 50 min, flow rate 15 mL/min,
l
l
lmol) in
dry methanol (1 mL) was added MSH(4) azide 5 (29 mg, 36
lmol).
The reaction mixture was subjected to microwave irradiation (Bio-
tage Initiator, 100 °C) for 4 h. The reaction mixture was diluted
with water (25 mL) and extracted (3 Â 30 mL) with a solution of
dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was
washed with CHCl3 (2 Â 20 mL) and lyophilized to give a white so-
lid. Purification by HPLC (stationary phase 19 Â 256 mm X-Bridge
Preparative C18 column, mobile phase 10–90% acetonitrile and
water containing 0.1% TFA within 50 min, flow rate 15 mL/min,
UV detection at 230 nm) yielded 13 mg (5 lmol, 40%) of 14b, tR
23.1–25.9 min; analytical HPLC (stationary phase 3 Â 150 mm
3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear
gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in
water and B is 0.1% TFA in acetonitrile, detection at 220 and
280 nm), tR 19.4 min (broad); HRMS (ESI) calcd for
UV detection at 230 nm) yielded 8 mg (5
lmol, 18%) of 13a, tR
C
146H228N34O28 (M + 4H)4+ 726.4360, obsd 726.4353.
21.6–24.9 min; analytical HPLC (stationary phase 3 Â 150 mm
3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear
gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in
water and B is 0.1% TFA in acetonitrile, detection at 220 and
280 nm), tR 20.2 min (broad); HRMS (ESI) calcd for C74H122N14O11
(M + 2H)2+ 691.4703, obsd 691.4699.
2.1.4.7. Synthesis and characterization of 15a.
of alkyne 9 (5 mg, 8 mol), TBTA (3 mg, 6 mol), and tetrakis(ace-
tonitrile)copper(I) hexafluorophosphate (2 mg, mol) in dry
methanol (1 mL) was added NDP- -MSH azide 7 (16 mg, 9 mol).
The reaction mixture was subjected to microwave irradiation (Bio-
tage Initiator, 100 °C) for 4 h. The reaction mixture was diluted
with water (25 mL) and extracted (3 Â 30 mL) with a solution of
dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was then
washed with CHCl3 (2 Â 20 mL) and lyophilized to give a white so-
lid. Purification by HPLC (stationary phase 19 Â 256 mm X-Bridge
Preparative C18 column, mobile phase 10–90% acetonitrile and
water containing 0.1% TFA within 50 min, flow rate 15 mL/min,
To a mixture
l
l
6
l
a
l
2.1.4.4. Synthesis and characterization of 13b.
of alkyne 10 (15 mg, 22 mol), TBTA (3 mg, 6
To a mixture
mol), and tetra-
l
l
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
lmol) in
dry methanol (1 mL) was added MSH(4) azide 5 (36 mg, 46
lmol).
The reaction mixture was subjected to microwave irradiation (Bio-
tage Initiator, 100 °C) for 4 h. The reaction mixture was diluted
with water (25 mL) and extracted (3 Â 30 mL) with a solution of
dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was
washed with CHCl3 (2 Â 20 mL) and lyophilized to give a white so-
lid. Purification by HPLC (stationary phase 19 Â 256 mm X-Bridge
Preparative C18 column, mobile phase 10–90% acetonitrile and
water containing 0.1% TFA within 50 min, flow rate 15 mL/min,
UV detection at 230 nm) yielded 11 mg (5 lmol, 58%) of 15a, tR
23.7–26.2 min; analytical HPLC (stationary phase 3 Â 150 mm
3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear
gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in
water and B is 0.1% TFA in acetonitrile, detection at 220 and
280 nm), tR 19.5 min (broad); HRMS (ESI) calcd for
UV detection at 230 nm) yielded 26 mg (11
lmol, 53%) of 13b, tR
C
118H191N24O25 (M + 3H)3+ 781.4799, obsd 781.4789.
18.2–22.6 min; analytical HPLC (stationary phase 3 Â 150 mm
3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear
gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in
water and B is 0.1% TFA in acetonitrile, detection at 220 and
280 nm), tR 18.6 min (broad); HRMS (ESI) calcd for
2.1.4.8. Synthesis and characterization of 15b.
of alkyne 10 (3.7 mg, 5 mol), TBTA (3 mg, 6
To a mixture
mol), and tetra-
l
l
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
dry methanol (1 mL) was added NDP- -MSH azide 7 (20 mg,
11 mol). The reaction mixture was subjected to microwave irradi-
lmol) in
a
C
118H181N28O16 (M + 3H)3+ 748.8064, obsd 748.8063.
l
ation (Biotage Initiator, 100 °C) for 4 h. The reaction mixture was
diluted with water (25 mL) and extracted (3 Â 30 mL) with a solu-
tion of dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was
then washed with CHCl3 (2 Â 20 mL) and lyophilized to give a
white solid. Purification by HPLC (stationary phase 19 Â 256 mm
X-Bridge Preparative C18 column, mobile phase 10–90% acetoni-
trile and water containing 0.1% TFA within 50 min, flow rate
2.1.4.5. Synthesis and characterization of 14a.
of alkyne 9 (10 mg, 17 mol), TBTA (3 mg, 6
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
To a mixture
mol), and tetra-
l
l
lmol) in
dry methanol (1 mL) was added MSH(7) azide 6 (20 mg, 18
lmol).
The reaction mixture was subjected to microwave irradiation (Bio-
tage Initiator, 100 °C) for 4 h. The reaction mixture was diluted
with water (25 mL) and extracted (3 Â 30 mL) with a solution of
dithizone (3 mg) in CHCl3 (150 mL). The aqueous phase was
washed with CHCl3 (2 Â 20 mL) and lyophilized to give a white so-
lid. Purification by HPLC (stationary phase 19 Â 256 mm X-Bridge
Preparative C18 column, mobile phase 10–90% acetonitrile and
water containing 0.1% TFA within 50 min, flow rate 15 mL/min,
15 mL/min, UV detection at 230 nm) yielded 13 mg (3 lmol, 62%)
of 15b, tR 22.1–24.9 min; analytical HPLC (stationary phase
3 Â 150 mm 3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/
min, linear gradient from 10–90% B in A over 45 min, where A is
0.1% TFA in water and B is 0.1% TFA in acetonitrile, detection at
220 and 280 nm), tR 17.9 min (broad); HRMS (ESI) calcd for
UV detection at 230 nm) yielded 7 mg (4
lmol, 24%) of 14a, tR
C
206H317N48O44 (M + 3H)3+ 1389.1342, obsd 1389.1344.
25.2–27.9 min; analytical HPLC (stationary phase 3 Â 150 mm
3.5 Å Waters C18 X-Bridge column, flow rate 0.3 mL/min, linear
gradient from 10–90% B in A over 45 min, where A is 0.1% TFA in
water and B is 0.1% TFA in acetonitrile, detection at 220 and
280 nm), tR 20.2 min (broad); HRMS (ESI) calcd for C88H146N17O17
(M + 3H)3+ 571.0355, obsd 571.0365.
2.1.4.9. Synthesis and characterization of 16 and 17.
Tria-
zole 16 was prepared from the corresponding resin-bound tetra-
peptide as previously described.20 Triazole 17 was prepared in a
similar manner from the corresponding resin-bound heptapeptide.
Following attachment of the N-terminal 6-(4-butyl-1H-1,2,3-tria-
zol-1-yl)hexanoic acid residue, cleavage and deprotection were
achieved using a 91:3:3:3 mixture of trifluoroacetic acid, triisopro-
pylsilane, thioanisole, and water (10 mL). The mixture of cleavage
cocktail and resin was shaken overnight, the solution was sepa-
rated from the resin, volatiles were evaporated, the residue tritu-
rated with ether, and the crude product separated by
centrifugation. Purification of 17 was accomplished by reversed
2.1.4.6. Synthesis and characterization of 14b.
of alkyne 10 (7.5 mg, 11 mol), TBTA (3 mg, 6
To a mixture
mol), and tetra-
l
l
kis(acetonitrile)copper(I) hexafluorophosphate (2 mg, 6
lmol) in
dry methanol (1 mL) was added MSH(7) azide 6 (26 mg, 23
lmol).
The reaction mixture was subjected to microwave irradiation (Bio-
tage Initiator, 100 °C) for 4 h. The reaction mixture was diluted
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6
R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
phase chromatography using 19 Â 256 mm X-Bridge Preparative
C18 column. The mobile phase was 10–90% acetonitrile and water
containing 0.1% TFA within 50 min; the flow rate was 15 mL/min
and the UV detector system operated at 230 nm. HRMS (ESI) calcd
for C58H85N17O11 (M + 2H)2+ 597.8301, obsd 597.8295.
methods.3,4,14,15 HEK-293 cells engineered to express both hMC4R
and hCCK2R were used to assess ligand binding.15 Cells were
grown in Dulbecco’s Modified Eagle Medium (DMEM) supple-
mented with 10% fetal bovine serum. Cells were seeded in Costar,
tissue culture treated, black frame with transparent well, 96-well
plates (part #3603) at a density of 2 Â 104 cells per well and were
incubated at 37 °C to reach 80–90% confluence. Ligands were di-
luted in binding buffer (1L DMEM, 5.97 g Hepes, 0.2% BSA, 1 mM
1,10-phenanthroline, 0.5 mg/L leupeptin, 200 mg/L bacitracin, pH
adjusted to 7.4 using 2 N NaOH). On the day of an experiment, se-
rial dilutions of Eu-labeled probes 2–4 (100 lL total volume) were
made in different well plates in preparation for addition to plates
containing cells. Plates containing cells were removed from the
incubator and media was removed. Binding buffer (50
was added to the first four rows of a plate and 25 M NDP-
MSH in binding buffer (50 L/well) was added to the next four
rows to determine non-specific binding. Fifty L of the solutions
lL/well)
l
a-
l
l
of pre-diluted probes 2–4 were then added to the cell-containing
plates and the plates were incubated at 37 °C. After 1 h the media
was removed and the plates were washed (3 Â 175
wash buffer (1L DMEM, 1 g BSA, 20 M EDTA, 0.01% Tween20).
Enhancement solution (PerkinElmer 1244–105) was added
lL/well) using
l
(105 lL/well) and the plates were incubated for 30 min at 37 °C be-
2.1.4.10. Synthesis and characterization of 18–20.
The prep-
fore fluorescence was measured using a VICTORTM X4 2030 Multi-
label Reader (PerkinElmer) employing the standard Eu time-
resolved fluorescence (TRF) measurement settings (340 nm excita-
aration and characterization of triazoles 18–20 was previously
described.17
tion, 400
l
s delay, and emission collection for 400
ls at 615 nm).
2.2.2.2. Competition Binding Assays.
Serial dilutions of com-
pounds to be tested were made in different well plates in binding
buffer (1L DMEM, 5.97 g Hepes, 0.2% BSA, 1 mM 1,10-phenanthro-
line, 0.5 mg/L leupeptin, 200 mg/L bacitracin, pH adjusted to 7.4
using 2 N NaOH, 50 lL total volume) before adding to plates con-
taining cells. Stock solutions of Eu-labeled probes were diluted
with binding buffer to a concentration of 40 nM for 3 and 10 nM
for 4. Plates containing cells were removed from the incubator,
media was removed and replaced with binding buffer containing
the Eu-labeled probe (50 lL/well), the solutions of compounds to
be tested (concentration range generally 10À4–10À11 M) were
added, and the plates incubated at 37 °C. After 1 h the media was
removed and the plates were washed (3 Â 175
buffer (1L DMEM, 1 g BSA, 20 M EDTA, 0.01% Tween20). Enhance-
ment solution (PerkinElmer 1244–105) was added (105 L/well)
lL/well) using wash
l
l
and the plates were incubated for 30 min at 37 °C before fluores-
cence was measured using using a VICTORTM X4 2030 Multilabel
Reader (PerkinElmer) employing the standard Eu time-resolved
fluorescence (TRF) measurement settings (340 nm excitation,
400
ls delay, and emission collection for 400
ls at 615 nm).
2.2.2.3. Data Analysis.
Binding data were analyzed using
GraphPad Prism software. Saturation binding data were analyzed
using nonlinear regression analysis and fitted to classic one site to-
tal binding and nonspecific binding equations. Results from satura-
tion binding experiments are depicted in Figure 2 and given in
Table 2. The values of Kd given represent averages of four indepen-
dent saturation binding experiments, each consisting of four data
points per concentration tested. Competitive binding data were
analyzed using nonlinear regression analysis and fitted to a classic
one site binding competition equation. Each IC50 value was gener-
ated from individual competitive binding assays and converted to a
Ki value using the equation Ki = IC50/(1 + ([ligand]/Kd)) where [li-
gand] refers to the concentration of the probe used as the labeled
competed ligand. For probe 3, [ligand] = 20 nM and Kd = 27 nM.
For probe 4, [ligand] = 5 nM and Kd = 4.2 nM. Results from compe-
tition binding experiments are given in Table 3. The values of Ki gi-
ven represent averages of four independent competition binding
2.2. Biological studies
2.2.1. Formulation of Solutions
Stock solutions of probes 2–4, squalene-derived constructs 12a-
15a and 12b-15b, and control compounds 16,20 17, and 7 were
made up in water. Concentrations were initially based on mea-
sured weights of solutes and were confirmed by UV analysis.
2.2.2. Binding Assays
2.2.2.1. Saturation Binding Assays.
binding assays were carried out following previously described
Quantitative receptor-
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R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Table 2
Reaction of squalene with excess disiamylborane, followed by
oxidation, produced hexaol 8 in 51% yield as a white foam
(Scheme 1). Analysis by 13C NMR (Fig. 1) confirmed that hydrobo-
ration had been regioselective and had yielded a mixture of diaste-
reomeric secondary alcohols as depicted in 8.16,20 Treatment of 8
with three equivalents of sodium hydride and five equivalents of
6-bromo-1-hexyne21 in DMF gave a mixture of alkylation products
from which monoalkynes 9 and dialkynes 10 were isolated in 21%
and 43% yields, respectively, by silica gel column chromatography.
It is assumed that 9 and 10 are mixtures of regioisomers due to
random alkylation at oxygen atoms along the carbon backbone.
Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reac-
tions23 of 9 and 10 with excesses of the serine amide-derived azide
1120 gave the corresponding triazole-containing products 12a and
12b in 52% and 90% yields, respectively, following chromato-
graphic purification (Scheme 1). These compounds were character-
ized by NMR and ESI MS. CuAAC reactions of 9 and 10 with
excesses of azide 5 gave the corresponding triazoles 13a and
13b. Copper ions were removed from the crude product mixtures
by complexation with dithizone and removal of the complex by
extraction with CHCl3.24 The water-soluble products 13a and 13b
were purified by preparative reversed phase HPLC, recovered by
lyophilization in 18% and 53% yields, respectively, and character-
ized by ESI MS. In a similar manner, CuAAC reactions of 9 and 10
with excesses of azides 6 and 7 gave the corresponding triazole-
containing products 14a, 14b, 15a, and 15b in 24%, 40%, 58%, and
62% yields, respectively, after workup and purification (Scheme 1).
These compounds were characterized by ESI MS. The synthesis and
characterization of compound 16 was previously reported.20
Compound 17 was prepared in a similar manner by N-terminal
acylation of the resin-bound heptapeptide with 6-(4-butyl-1H-
1,2,3-triazol-1-yl)hexanoic acid.20 The synthesis and characteriza-
tion of compounds 18–20 were previously reported.17
Binding constants for Eu-DTPA-PEGO probes 2–4 with hMC4R.
a
Probe
Kd
2
3
4
93 100 nMb
27 3.9 nMc
4.2 0.48 nMd
a
The value given is the average of four independent binding experiments, each
done in quadruplicate.
b
Includes data up to [2] = 400 nM.
Includes data up to [3] = 150 nM.
Includes data up to [4] = 30 nM.
c
d
experiments, each consisting of four data points per concentration
tested.
3. Results
3.1. Chemistry
Fully protected resin-bound precursors to probes 2–4 and
azides 5–7 were prepared by solid phase synthesis on Rink amide
Tentagel S resin (for probe synthesis) or on Rink amide resin (for
ligand synthesis). For the preparation of probes 2–4, Fmoc-PEGO18
was coupled to the N-terminus of the resin-bound peptides. Fol-
lowing removal of the Fmoc, DTPA,19 activated 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
produced the metal-free precursors to 2–4 which were purified by
preparative HPLC and characterized by FT-ICR MS. Following com-
plexation of Eu3+, the Eu-DTPA-PEGO probes 2–4 were purified by
reversed phase chromatography and characterized by analytical
HPLC and FT-ICR MS (Table 1).
For the preparation of azides 5–7, 6-azidohexanoic acid22 was
coupled to the N-terminus of the resin-bound peptides. Simulta-
neous side chain deprotection and cleavage of the peptide from
the resin produced compounds 5–7 which were purified by
reversed phase preparative HPLC and characterized by ESI MS
(Table 1). Yields ranged from 39–60%.
3.2. Bioassays
HEK-293 cells overexpressing both hMC4R8,25 and hCCK2R26
were used to measure the affinities of probes 2–4 for binding to
hMC4R by means of saturation binding assays (Fig. 2).15 Kd values
for probes 2–4 are given in Table 2. Europium-based time-resolved
fluorescence competitive binding assays using Eu-DTPA-PEGO-
MSH(7) (3) and Eu-DTPA-PEGO-NDP-
peted probes were employed to study the binding of monovalent
and bivalent serinamide, MSH(4), MSH(7), and NDP- -MSH con-
structs 12a-15a and 12b-15b. Competitive binding assays using
probe 3 as the competed probe were employed to study the bind-
ing of monovalent, divalent, and trivalent MSH(4) constructs 18–
20.17 Ki values for these compounds and for the control compounds
16, 17, and 7 are given in Table 3.
a-MSH-NH2 (4) as the com-
Table 3
Competitive binding of Eu-DTPA-PEGO probes 3 and 4 against monovalent constructs
12a-15a, bivalent constructs 12b-15b, monovalent, divalent, and trivalent com-
pounds 18–20, and control compounds 16, 17, and 7 with hMC4R.
a
Compounds
Probe
Probe
3Kia,b (nM)
4Kia,c (nM)
12a
12b
16
13a
13b
17
14a
14b
7
15a
15b
18
ncbd
ncbd
ncbd
ncbd
350 28
600 16
150 38
1.8 0.2
2.4 0.8
1.7 0.2
3.1 0.4
6.0 0.2
1.5 0.9
330 52
37 10
4.3 0.4
990 40
2200 150
810 56
18 1.0
33 0.6
15 1.5
0.14 0.01
15 1.8
3.4 1.4
3900 600e
250 58e
11 1.2e
4. Discussion
Previously we described the preparation and testing of scaffolds
derived from squalene,14,16 solanesol,20 and sucrose27 with one or
more sidechains bearing MSH(4), a ligand with a low micromolar
affinity for binding to hMC4R. MSH(4) was chosen because syner-
gistic effects are generally more easily detected for multivalent
constructs of low-affinity ligands.9–13 All of the monovalent and
multivalent MSH(4) constructs derived from squalene, solanesol,
and sucrose exhibited competitive binding to hMC4R that was
comparable to the parental ligand. The competed probe used in
19
20
a
The Ki was calculated using the equation Ki = IC50/(1+([ligand]/Kd)), where
[ligand] refers to the concentration of the probe used as the labeled competed
ligand.
these studies was Eu-DTPA-NDP-
a-MSH (1), which is based on
b
Here [ligand] = 20 nM, the concentration of probe 3.
Here [ligand] = 5 nM, the concentration of probe 4.
ncb = no competitive binding.
Calculated from EC50 values taken from reference 17, wherein a probe similar to
the superpotent ligand NDP-
a
-MSH. The off-rate of NDP- -MSH
a
c
is approximately eight hours.28 In addition, evidence suggests that
receptor-bound 1 is taken up by the cells.29 For these reasons, it
was clear that competition binding assays involving 1 and hMC4R
d
e
4 was employed.
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8
R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Scheme 1. Synthesis of monovalent constructs 12a–15a and divalent constructs 12b–15b. Compounds 8, 9, and 10 were obtained as complex mixtures of stereoisomers and
regioisomers. Compounds 12a–15a and 12b–15b, derived from 9 and 10, respectively, are likewise assumed to be the related mixtures of stereoisomers and regioisomers.
on living cells do not take place under thermodynamic control. This
fact raised the possibility that the competition for binding to
hMC4R between 1 and any variant of MSH(4) was biased in favor
of 1, to the detriment of multivalent binding.
dent binding and/or uptake as opposed to receptor saturation.
However, at sufficiently low concentrations, the differences be-
tween total and non-specific binding for probes 3 and 4 were sig-
nificant, and permitted the calculation of reasonable Kd values (see
Table 2) so that use of these probes in competition binding assays
was deemed possible.
For competition assays, a range of ligand potencies attached to
monovalent, bivalent, and trivalent constructs was desired. Azides
5–7 were prepared by standard solid phase methods. Scaffolds 9
and 10 were prepared from squalene via hexaol 8 as depicted in
Scheme 1.16 Attachment of azides 5–7 to 9 and 10 by means of
microwave-assisted copper(I)-catalyzed azide-alkyne cycloaddi-
tion (CuAAC)23 produced monovalent and bivalent constructs 13a
and 13b incorporating MSH(4), 14a and 14b incorporating
To determine what effects might result from the use of probes
with a lesser affinity for hMC4R, the set of Eu-DTPA-PEGO probes
2–4 was prepared by standard solid phase methods. Saturation
binding assays for 2–4 were carried out using HEK-293 cells that
stably overexpress both hMC4R (6 Â 105 receptors per cell) and
hCCK2R (1 Â 106 receptors per cell).15 These cells were employed
so that results obtained here will be comparable with results from
studies with multimeric constructs that bear ligands targeted to
hCCK2R and with multimeric constructs that bear mixtures of li-
gands targeted to both receptors.30 In all cases, non-specific bind-
ing was determined by blocking binding to hMC4R with a high
MSH(7), and 15a and 15b incorporating NDP-a-MSH. Control com-
concentration of NDP-
a
-MSH. Although probe 2 exhibited 20% as
pounds 12a and 12b were prepared by CuAAC reaction of serine
amide-derived azide 1120 with 9 and 10. In addition, MSH(4)-con-
taining compounds 18–20, which were recently shown to exhibit
enhanced avidity with increased ligand number when competed
much non-specific binding as did probes 3 and 4 (see Fig. 2), the
difference between total binding and non-specific binding for 2
was small, even at very low concentrations of the probe. Thus,
the measured Kd has a large associated error (93 100 nM), and
against an NDP-a
-MSH-based probe,17 were here competed
plans to use probe
2
in competition binding assays were
against MSH(7)-based probe 3. Compounds 16,20 17, and 7 were
used as reference compounds in place of the parental ligands.
Results of the competition binding assays are given in Table 3.
The serine amide-containing control compounds 12a and 12b were
both ineffective at blocking probes 3 and 4 from binding to hMC4R
abandoned.31
As with probe 2, graphs of total and non-specific binding for
probes 3 and 4 appear to be linear and parallel at high concentra-
tions (see Fig. 2), consistent with some form of receptor-indepen-
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R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
Figure 1. Top: A portion of the DEPT 135 13C NMR spectrum of 8 in CD3OD. Bottom: A portion of the 13C NMR spectrum of 8 in CD3OD. The lines near 73 ppm are due to
tertiary carbons bearing OH groups and the lines between 76–79 ppm are due to secondary carbons bearing OH groups. The spectral complexity occurs because 8 is a mixture
of regioisomers and stereoisomers.
Probe 2
Probe 3
Probe 4
Figure 2. Saturation binding curves for Eu-DTPA-PEGO probes 2–4. Total binding (d). Non-specific binding (j). Specific binding (N). The graphs in the right-hand column are
truncated expansions of the graphs in the left-hand column and include a specific binding curve.
over the range of concentrations tested. For the squalene-derived
monovalent and divalent constructs, the trends in Ki values are
consistent for all three ligands tested, MSH(4) (compare 16, 13a,
and 13b), MSH(7) (compare 17, 14a, and 14b), and NDP-a-MSH
(compare 7, 15a, and 15b). Squalene-derived monovalent com-
of two, presumably due to compromised ligand binding, while
the corresponding divalent compound exhibits a lower Ki value, of-
ten a factor of two that can be ascribed to statistical doubling of the
number of ligands. In all but one case, the Ki value obtained using
probe 4 was higher than the corresponding Ki value obtained using
probe 3, a result that is consistent with the stronger binding of 4
pounds exhibit a higher Ki value than the control, often a factor
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10
R. Alleti et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
6. Castrucci, A. M. L.; Hadley, M. E.; Sawyer, T. K.; Wilkes, B. C.; Al-Obeidi, F.;
Staples, D. J.; de Vaux, A. E.; Dym, O.; Hintz, M. F.; Riehm, J. P.; Rao, K. R.; Hruby,
V. J. Gen. Comp. Endocrinol. 1989, 73, 157.
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V. J.; Dickinson, C.; Gantz, I. J. Med. Chem. 1997, 40, 2133.
8. The hMC4R vector was originally received from Dr. Ira Gantz; see Gantz, I.;
Miwa, H.; Konda, Y.; Shimoto, Y.; Tashiro, T.; Watson, S. J.; DelValle, J.; Yamada,
T. J. Biol. Chem. 1993, 268, 15174.
9. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem. Int. Ed. 1998, 37,
2754.
10. Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Angew. Chem. Int. Ed. 2006, 45, 2348.
11. Krishnamurthy, V. M.; Estroff, L. A.; Whitesides, G. M. In Fragment-based
Approaches in Drug Discovery; Jahnke, W., Erlanson, D. A., Eds.; Wiley-VCH:
Weinheim, 2006; pp 11–53.
12. Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. ACS
Chemical Biology 2007, 2, 119.
versus 3 to hMC4R. In view of past results with monovalent and
multivalent compounds derived from squalene,14,16 solanesol,20
sucrose,27 and compounds 18–20,17 the results herein suggest that
the MSH(4) ligands in compounds 13b-15b are spaced too far apart
for manifestation of multivalent binding.
Enhanced avidity interpretable as multivalent binding was ob-
served when compounds 18–20 were competed against probe 3,
although the lowering of the Ki values with each additional ligand
(approximately ninefold at each step) was attenuated when
compared with Ki values calculated from published EC50 data for
competition binding assays that employed a Eu-DTPA-PEGO-NDP-
a
-MSH probe similar to 4 (approximately 16-fold and 22-fold, see
Table 3).17
Although different sets of data were produced, similar conclu-
13. Choi, S.-K. Synthetic Multivalent Molecules; Wiley-Interscience: Hoboken, NJ,
2004.
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.
15. Xu, L.; Vagner, J.; Josan, J. S.; 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. Cells with a
passage number less than 50 were used in assays.
16. Jagadish, B.; Sankaranarayanan, R.; Xu, L.; Richards, R.; Vagner, J.; Hruby, V. J.;
Gillies, R. J.; Mash, E. A. Bioorg. Med. Chem. Lett. 2007, 17, 3310.
17. Brabez, N.; Lynch, R. M.; Xu, L.; Gillies, R. J.; Chassaing, G.; Lavielle, S.; Hruby, V.
J. J. Med. Chem. 2011, 54, 7375.
sions were reached from competition binding experiments that
employed probes 3 and 4, regardless of the potency of the ligand
being competed. Probe 3 requires less time and effort to prepare,
and so may find favor over probe 4 in future competition binding
studies. However, confirmation of observed binding behaviors by
the use of both probes would seem a prudent practice.
18. Jolimaitre, P.; Poirier, C.; Richard, A.; Blanpain, A.; Delord, B.; Roux, D.; Bourel-
Bonnet, L. Eur. J. Med. Chem. 2007, 42, 114. 1-(9H-fluoren-9-yl)-3,19-dioxo-
Acknowledgments
2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic
acid
(Fmoc-PEGO)
is
commercially available from Novabiochem as O-(N-Fmoc-3-aminopropyl)-O’-
(N-diglycolyl-3-aminopropyl)diethyleneglycol.
The authors thank Renata Patek for laboratory assistance. This
work was supported by grants R33 CA 95944, RO1 CA 97360,
RO1 CA 123547, and P30 CA 23074 from the National Cancer
Institute.
19. Diethylenetriamine pentaacetic acid dianhydride is commercially available.
20. Alleti, R.; Rao, V.; Xu, L.; Gillies, R. J.; Mash, E. A. J. Org. Chem. 2010, 75, 5895.
21. Sharma, S.; Oehlschlager, A. C. J. Org. Chem. 1989, 54, 5064.
22. Grandjean, C.; Boutonnier, A.; Guerreiro, C.; Fournier, J.-M.; Mulard, L. A. J. Org.
Chem. 2005, 70, 7123.
23. Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302.
24. Caplin, S. Tissue Culture Association Manual 1976, 2, 439.
25. Yang, X.; Wang, Z.; Dong, W.; Ling, L.; Yang, H.; Chen, R. J. Prot. Chem. 2003, 22,
335.
26. Noble, F.; Wank, S. A.; Crawley, J. N.; Bradwejn, J.; Seroogy, K. B.; Hamon, M.;
Roques, B. P. Pharmacol. Rev. 1999, 51, 745.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.052.
27. Rao, V.; Alleti, R.; Xu, L.; Tafreshi, N. K.; Morse, D. L.; Gillies, R. J.; Mash, E. A.
Bioorg. Med. Chem. 2011, 19, 6474–6482.
References and notes
28. Haskell-Luevano, C.; Miwa, H.; Dickinson, C.; Hadley, M. E.; Hruby, V. J.;
Yamada, T.; Gantz, I. J. Med. Chem. 1996, 39, 432.
29. Xu, L. Unpublished work.
30. Josan, J. S.; Handl, H. L.; Sankaranarayanan, R.; Xu, L.; Lynch, R. M.; Vagner, J.;
Mash, E. A.; Hruby, V. J.; Gillies, R. J. Bioconjugate Chem. 2011, 22, 1270–1278.
1. Sawyer, T. K.; Sanfilippo, P. J.; Hruby, V. J.; Engel, M. H.; Heward, C. B.; Burnett, J.
B.; Hadley, M. E. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 5754.
2. Hadley, M. E.; Anderson, B.; Heward, C. B.; Sawyer, T. K.; Hruby, V. J. Science
1981, 213, 1025.
3. Handl, H. L.; Vagner, J.; Yamamura, H. I.; Hruby, V. J.; Gillies, R. J. Anal. Biochem.
2004, 330, 242.
31. In a prior study (see reference 14), a Kd of 9.1 1.4 lM was measured for probe
2, and much less non-specific binding relative to total binding was observed.
4. De Silva, C. R.; Vagner, J.; Lynch, R.; Gillies, R. J.; Hruby, V. J. Anal. Biochem. 2010,
398, 15.
5. Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.; Sawyer, T. K.; Staples, D.
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The study employed HEK-293 cells that transiently overexpressed only hMC4R
(6 Â 105 receptors per cell), a large excess of MSH(4) instead of NDP-
a-MSH as
the blocking agent in the determination of non-specific binding, and a different
brand of 96-well plates. It is unclear which factors, if any, account for the
different outcome for probe 2 in the present study.
Please cite this article in press as: Alleti, R.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.052