A sucrose-derived scaffold for multimerization of bioactive peptides

Article abstract of DOI:10.1016/j.bmc.2011.08.053

A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N₃(CH₂)₅(C=O)-His-DPhe-Arg-Trp- NH₂ (MSH4) or N

Full text of DOI:10.1016/j.bmc.2011.08.053

Bioorganic & Medicinal Chemistry 19 (2011) 6474–6482
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A sucrose-derived scaffold for multimerization of bioactive peptides
Venkataramanarao Rao a, Ramesh Alleti a, Liping Xu b, Narges K. Tafreshi b, David L. Morse b,
Robert J. Gillies b, Eugene A. Mash a,
⇑
a Department of Chemistry and Biochemistry, The University of Arizona, Tucson, AZ 85721-0041, USA
b Department of Functional and Molecular Imaging, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from
sucrose. One or more copies of a tetrapeptide azide, either N3(CH2)5(C@O)-His-DPhe-Arg-Trp-NH2
(MSH4) or N3(CH2)5(C@O)-Trp-Met-Asp-Phe-NH2 (CCK4), were attached to the scaffold via the
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using
Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-
Received 23 June 2011
Revised 18 August 2011
Accepted 24 August 2011
Available online 27 August 2011
Pro-Val-NH2 (NDP-a-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) were used to study the
Keywords:
interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered
to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited bind-
ing comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate
for multivalent binding, or MSH4 is too weak a binder for a second ‘anchoring’ binding event to occur
before the monovalently-bound construct is released from the cell surface. In contrast with this behavior,
monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent
CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multi-
valent binding, which may be due to increased residence times for monovalently bound CCK4 constructs
on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the estab-
lishment of multivalent binding.
Multimeric ligands
Melanocortin 4 receptor ligands
Cholecystokinin 2 receptor ligands
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
enhanced affinity and selectivity for cancer cells based on cooper-
ative binding.12–16
Early detection and diagnosis of many human cancers could be
aided by reagents that seek out and selectively bind to cancer cells
and report their existence and location by non-invasive molecular
imaging.1–4 One strategy for development of such reagents in-
volves attaching imaging agents to molecular scaffolds that bear
multiple copies of ligands to receptors present on the surface of
cancer cells.5–11 Such multivalent constructs could display
Previously we described the preparation and testing of multiva-
lent constructs derived from squalene17 and solanesol.18 These
constructs bore sidechains based on the ligand Ac-His-DPhe-Arg-
Trp-NH2 (MSH4)19–21 which has a low micromolar affinity for
binding to the melanocortin 4 receptor (MC4R).22 In our prior
work, the azide N3(CH2)5(C@O)-His-DPhe-Arg-Trp-NH2 (1, Scheme
1) was attached to a linear scaffold (e.g., 2) bearing terminal alkyne
groups using the copper(I)-catalyzed azide-alkyne cycloaddition
(CuAAC),23–26 producing triazole-containing multivalent con-
structs (e.g., 3).18 The abilities of 3 and of similar monovalent
and multivalent constructs based on MSH4 to bind to MC4R were
tested in competitive binding assays against probes 411 and 527
using Hek293 cells engineered to overexpress this receptor. Probe
4 is based on the superpotent ligand Ser-Tyr-Ser-Nle-Glu-His-
Abbreviations: Boc, tert-butoxycarbonyl; BSA, bovine serum albumin; CCK2R,
cholecystokinin
2 receptor; CCK4, Trp-Met-Asp-Phe-NH2; Cl-HOBt, 6-chloro-1-
hydroxybenzotriazole; CuAAC, copper(I)-catalyzed azide-alkyne cycloaddition;
DCM, dichloromethane; DIC, diisopropyl carbodiimide; DMEM, Dulbecco’s Modified
Eagle Medium; DMF, N,N-dimethylformamide; DTPA, diethylenetriaminepentaace-
tic acid; EC50, effective concentration, 50%; ESI, electrospray ionization; Fmoc, 9-
fluorenylmethyoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HRMS, high resolution
mass spectroscopy; MALDI–TOF, matrix-assisted desorption/ionization time-of-
flight; MC4R, melanocortin 4 receptor; MEM, minimum essential medium; MSH4,
DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-a
-MSH),28,29 while the
probe 5 is based on MSH4. Interestingly, all of the solanesol-de-
rived monovalent and multivalent constructs bearing MSH4 bound
monovalently to MC4R when competed against either probe.18 To
determine whether or not the solanesol-derived linear scaffold
was responsible for this unexpected binding behavior, we have
prepared and tested monovalent and multivalent constructs using
Ac-His-DPhe-Arg-Trp-NH2; NDP-a-MSH, Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-
Gly-Lys-Pro-Val-NH2; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; t-
Bu, tert-butyl; 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@u.arizona.edu (E.A. Mash).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.053

V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
6475
Scheme 1. Synthesis of multivalent constructs 3.
Scheme 2. Synthesis of sucrose derivative 9.
azide 1 and a spherical scaffold derived from sucrose (vide infra).
To determine whether or not this binding behavior was ligand
and/or receptor dependent, we have also prepared and tested
monovalent and multivalent constructs derived from this spherical
scaffold and an azide that incorporates the ligand Trp-Met-Asp-
Phe-NH2 (CCK4) which has a low nanomolar affinity for binding
to the cholecystokinin 2 receptor (CCK2R).30
While searching for alternatives to linear scaffolds for ligand
display, our attention was drawn by Olestra, a non-digestible fat
substitute (e.g., 6, Fig. 1).31,32 We reasoned that sucrose-derived
molecules might make useful scaffolds for multimerization if
ligands and other moieties of interest, such as imaging and thera-
peutic agents, could be attached to the termini of the fatty acid
chains. Additionally, we determined that the 1,4-disubstituted-

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V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
exposure, by exposure to I2 vapor, or by staining with 10% phos-
phomolybdic acid solution in ethanol or with 5% H2SO4 in ethanol
and heat. Gravity-driven column chromatography was accom-
plished using Silica Gel 60 (63–210 l
m). 1H NMR and 13C 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 ex-
pressed in ppm and are internally referenced (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). Analytical HPLC was per-
formed on a 4.6 ꢀ 75 mm Waters SymmetryÒ C18 column and pre-
parative HPLC was performed on a 19 ꢀ 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 rates were 1 mL/min and 15 mL/min for analytical and
preparative runs, respectively. The dual UV detector system oper-
ated at 230 and 280 nm. Matrix-assisted desorption/ionization
time-of-flight (MALDI–TOF) experiments were carried out on a
Bruker Ultraflex III MALDI TOF-TOF instrument. Both the reflectron
and linear techniques were used for positive ion detection. The ma-
trix, sinapic acid, and the analyte were dissolved in water/acetoni-
trile 1:1 containing 0.1% formic acid and the solutions mixed in a
ratio of 100:1. ESI was also used to ionize some of the samples.
These experiments were performed on an ESI Bruker Apex Qh 9.4
T FT-ICR instrument using standard ESI conditions. The samples
were dissolved in water/acetonitrile 1:1 containing 0.1% formic
acid in a concentration range of 1-30 lM.
Figure 1. A space-filling representation of one component of Olestra (6). This
representation Ó 1997 by Daniel J. Berger and may be copied without limit if its use
is for non-profit educational purposes.
2.1.2. Scaffold synthesis (Scheme 2)
2.1.2.1. Octa-O-(5-hexyn-1-yl)-b-
D-fructofuranosyl-a-D-glucopy-
ranoside (9). To a suspension of NaH (330 mg, 13.7 mmol) in
dry dimethylformamide (DMF, 10 mL) under argon were added
sucrose (200 mg, 0.58 mmol), 6-bromo-1-hexyne33 (1.13 g, 6.97
mmol), and tetrabutylammonium bromide (50 mg, 0.15 mmol).
The mixture was stirred at room temperature for 48 h. The reaction
was quenched with satd NH4Cl and the mixture extracted with
ethyl acetate (3 ꢀ 15 mL). The combined organic extracts were
washed with water, brine, dried over Na2SO4, and filtered. Removal
of volatiles in vacuo afforded a residue that was subjected to silica
gel chromatography (63–210 lm) using ethyl acetate/hexanes
(2:8) as elutant. This afforded 250 mg (0.25 mmol, 43%) of 9 as a
colorless oil, Rf 0.6 (ethyl acetate/hexanes, 3:7), ½a D25
ꢁ
17.0 (c 0.5,
CHCl3); IR (cmꢂ1) 3308, 2928, 2854, 1213, 1152, 1094; 1H NMR
(500 MHz, CDCl3) d 1.57–1.71 (m, 32H), 1.95 (m, 8H), 2.18–2.23
(m, 16H), 3.17 (dd, J = 9.5 Hz, J = 3.4 Hz, 1H), 3.28 (t, J = 9.5 Hz,
1H), 3.35–3.71 (m, 20H), 3.77–3.92 (m, 5H), 4.07 (d, J = 7.2 Hz,
1H), 5.50 (d, J = 3.8 Hz, 1H); 13C NMR (125 MHz, CDCl3) d 18.3,
24.7, 25.3, 28.6, 28.7, 29.1, 29.2, 29.6, 31.7, 62.3, 68.4, 68.5, 69.5,
70.2, 70.5, 70.6, 70.8, 71.0, 71.1, 71.7, 72.3, 72.4, 72.7, 79.5, 80.5,
81.6, 82.9, 84.0, 84.2, 84.3, 89.9, 104.3; HRMS (MALDI–TOF) calcd
for C60H86NaO11 [M+Na]+ 1005.6068, observed 1005.6068.
Figure 2. Superposition of cis-3-pentene (7) and 1,4-dimethyl-1H-1,2,3-triazole (8).
The methyl carbon-to-methyl carbon distances are 6.04 Å and 5.00 Å for 7 and 8,
respectively. The conformers of 7 and 8 used in this comparison were generated
using Spartan ‘02 v1.0.5 for Macintosh.
1H-1,2,3-triazole groups that are produced via CuAAC map
reasonably well onto the cis-1,2-disubstituted alkene groups of
unsaturated fatty acid components of Olestra (compare 7 and 8,
Fig. 2). These considerations led to the synthesis and biological
testing of sucrose-derived multivalent constructs that display one
or more copies of MSH4 or CCK4 as described herein.
2.1.3. Solid phase synthesis (Scheme 3)
In a syringe (polypropylene reaction tube equipped with a poly-
propylene frit) Rink amide resin (1 g, 0.68 mmol) was allowed to
swell in THF for 1 h. THF was removed, and 20% piperidine in DMF
(15 mL) was added for 2 min to cleave the 9-fluorenylmethyoxycar-
bonyl (Fmoc) group. The liquid phase was removed, and 20% piper-
idine solution in DMF (15 mL) was again added and the mixture
shaken for 18 min. The liquid phase was removed, and the resin
was washed with DMF (3 ꢀ 15 mL), DCM (3 ꢀ 15 mL), DMF
(3 ꢀ 15 mL), 0.5 M 1-hydroxybenzotriazole (HOBt) in DMF
(15 mL), 0.5 M HOBt in DMF (15 mL) plus a drop of bromophenol
blue, DMF (2 ꢀ 15 mL), and DCM (15 mL), in that order. A solution
of the first Fmoc-amino acid, Fmoc-Phe (1.05 g, 2.04 mmol),
6-chloro-1-hydroxybenzotriazole (Cl-HOBt, 345 mg, 2.04 mmol),
and diisopropylcarbodiimide (DIC, 512 mg, 4.08 mmol) in DMF
2. Materials and methods
2.1. Chemical synthesis
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
pre-coated Silica Gel 60 F-254 plates with visualization by UV

V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
6477
Table 1
trile)copper(I) hexafluorophosphate (1.4 mg, 3.8
l
mol) in dry meth-
Mass spectral and HPLC characterization of compounds 11 and 12
anol (1.5 mL) was irradiated for 2 h in a Biotage microwave reactor
(100 °C). After the reaction was complete, water (25 mL) was added
and the mixture extracted with CHCl3 containing dithizone (20 mg/
L, 3 ꢀ 15 mL) and with DCM (3 ꢀ 15 mL) to remove copper,35 TBTA,
and excess 9. After lyophilization, the residue (30 mg) was taken up
a
Compound Formula [M] Calcd mass [Ion] Mass found (error)
tR
11
12
C36H47N9O7
44H60N14O5 800.4454 [M+1]+ 800.4450 (0.5 ppm) 24.16
718.3671 [M+1]+ 718.3669 (0.3 ppm) 23.52
C
a
Linear gradient of from 10?90% acetonitrile in water containing 0.1% TFA over
in dry methanol (1.5 mL), 1-azidohexane (97 mg, 763
(6.3 mg, 11.8 mol), and tetrakis(acetonitrile)copper(I) hexafluoro-
phosphate (4.4 mg, 11.8 mol) were added, and the reaction mix-
lmol), TBTA
50 min.
l
l
(15 mL) was allowed to react for 2 min, then added to the resin and
the mixture shaken for 1 h, at which time the blue color had disap-
peared. The resin was then washed with DMF (3 ꢀ 15 mL), DCM
(3 ꢀ 15 mL), and DMF (3 ꢀ 15 mL). Free NH2 groups were capped
by addition of a 1:1 mixture of acetic anhydride and pyridine
(6 mL). After the mixture was shaken for 20 min, the resin was
washed with DMF (3 ꢀ 15 mL), DCM (3 ꢀ 15 mL), and DMF
(3 ꢀ 15 mL). The absence of free amine groups was confirmed by
the Kaiser test. The above cycle of procedures was repeated for cou-
pling of each of the amino acids in the sequence, and finally for
attachment of the N-terminal 6-azidohexanoic acid34 residue or
the N-terminal 6-(4-butyl-1H-1,2,3-triazol-1-yl)hexanoic acid18
residue, thus producing the resin-bound peptide derivatives related
to compounds 11 and 12. Cleavage and deprotection were achieved
using a 91:3:3:3 mixture of trifluoroacetic acid, triisopropylsilane,
thioanisole, and water (10 mL). The mixture of cleavage cocktail
and resin was shaken for overnight, the solution was separated from
the resin, volatiles were evaporated, the residue triturated with
diethyl ether, and the crude product collected by centrifugation.
Purification of the tetrapeptide products 11 and 12 was accom-
plished by preparative reversed phase HPLC. Yields ranged from
39% to 46% over several batches. The purity of compounds 11 and
12 was checked by analytical reversed phase HPLC. Compounds
11 and 12 were recovered from solution by lyophilization and were
further characterized by ESI mass spectrometry (see Table 1).
ture was irradiated for 4 h in a Biotage microwave reactor (100 °C).
Following workup as above and lyophilization, the residue was frac-
tionated by preparative HPLC (10?90% acetonitrile in water con-
taining 0.1% TFA within 50 min, tR 29–33 min) to afford 14a as an
oil; yield 13 mg (4.9 lmol, 26%). Product 14a was analyzed by MAL-
DI-TOF (see Fig. S2 in the Supplementary data) and by UV
spectroscopy.18
2.1.4.3. Procedure for reaction of azide-functionalized MSH4
derivative 1 and azide-functionalized serine amide derivative
10 with 9 toproduce multivalent constructs 14b.
of 9 (56 mg, 56 mol), TBTA (2.0 mg,
mol), azide 118 (15 mg, 19
3.8 mol), and tetrakis(acetonitrile)copper(I) hexafluorophosphate
(1.4 mg, 3.8 mol) in dry methanol (1.5 mL) was irradiated for 2 h
A mixture
l
l
l
l
in a Biotage microwave reactor (100 °C). After the reaction was
complete, water (25 mL) was added and the mixture extracted with
CHCl3 containing dithizone (20 mg/L, 3 ꢀ 15 mL) and with DCM
(3 ꢀ 15 mL) to remove copper,35 TBTA, and excess 9. After lyophili-
zation, the residue (29 mg) was taken up in dry methanol (1.5 mL),
azide 1018 (54 mg, 224
lmol), TBTA (6.9 mg, 3.8
lmol), and tetra-
kis(acetonitrile)copper(I) hexafluorophosphate (4.8 mg, 3.8
lmol)
were added, and the reaction mixture was irradiated for 4 h in a
Biotage microwave reactor (100 °C). Following workup as above
and lyophilization, the residue was fractionated by preparative
HPLC (10?90% acetonitrile in water containing 0.1% TFA within
50 min, tR 12.5–16.5 min) to afford 14b as a white powder; yield
2.1.4. Multimer synthesis (Scheme 4)
3.0 mg (0.87 lmol, 5%). Product 14b was analyzed by MALDI-TOF
2.1.4.1. Serine amide multimer 13. A mixture of 9 (10 mg,
(see Fig. S3 in the Supplementary data) and by UV spectroscopy.18
10
triazol-4-yl)methyl]amine (TBTA, 4.3 mg,
kis(acetonitrile)copper(I) hexafluorophosphate (3 mg, 8
l
mol), azide 1018 (39 mg, 162
l
mol), tris[(1-benzyl-1H-1,2,3-
mol), and tetra-
mol) in
8
l
2.1.4.4. General procedure for reaction of azide-functionalized
l
MSH4 derivative 1 and
derivative 10 with to
14c–e.
118 (variable amount, see Table 2), TBTA (0.43 mg/
tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.3 mg/
azide-functionalized serine amide
produce multivalent constructs
dry methanol (1.5 mL) was irradiated for 4 h in a Biotage micro-
wave reactor (100 °C). After the reaction was complete, water
(25 mL) was added and the mixture was extracted with CHCl3
containing dithizone (20 mg/L, 3 ꢀ 15 mL) to remove copper.35 The
water layer was then washed with DCM (2 ꢀ 15 mL) to remove any
remaining 10 and TBTA. After lyophilization, fractionation of the
residue by preparative HPLC (10?90% acetonitrile in water
containing 0.1% TFA within 50 min, tR 11.1 min) and recovery by
9
Mixtures of 9 (variable amount, see Table 2), azide
mol of 9), and
mol
mol of 9) were irradiated for 2–
l
l
of 9) in dry methanol (100 lL/l
4 h in a Biotage microwave reactor (100 °C). Azide 1018 (variable
amount, see Table 2) was then added to the reaction mixtures
and irradiation was resumed for another 4 h. After the reactions
lyophilization afforded 4.5 mg (1.5
l
mol, 15% yield) of 13 as a
were complete, water (2.5 mL/lmol of 9) was added and the mix-
white solid, mp 72–73 °C, ½a D25
ꢁ
6.5 (c 0.55, CHCl3); IR (cmꢂ1) 3283,
tures were extracted with CHCl3 containing dithizone (20 mg/L,
3 ꢀ 15 mL) and with DCM (3 ꢀ 15 mL) to remove copper,35 TBTA,
and excess 10. After lyophilization, the residues were fractionated
by preparative HPLC (10?90% acetonitrile in water containing
0.1% TFA within 50 min, tR 12.5–16.5 min) to afford products
2926, 2852, 1635, 1212, 1152; 1H NMR (500 MHz, CD3OD) d 1.31–
1.36 (m, 16H), 1.57–1.70 (m, 48H), 1.88–191 (m, 16H), 2.27 (t,
J = 7.0 Hz, 16H), 2.68–2.72 (m, 16H), 3.06–3.09 (m, 1H), 3.15–3.20
(m, 1H), 3.33–3.95 (m, 43H), 4.05 (d, J = 7.5 Hz, 1H), 4.33–4.42 (m,
23H), 5.50 (d, J = 3.5 Hz, 1H), 7.81 (br s, 8H); 13C NMR (125 MHz,
CD3OD) d 24.6, 25.6, 25.7, 25.8, 28.8, 29.2, 29.5, 35.1, 49.9, 55.1,
61.8, 66.2, 68.0, 69.6, 70.0, 70.2, 70.4, 70.6, 70.9, 71.0, 71.7, 71.9,
72.2, 72.5, 77.9, 79.2, 80.3, 81.3, 82.0, 83.7, 89.3, 92.5, 104.1, 109.4,
111.5, 113.4, 115.8, 122.1, 122.2, 147.3, 173.7, 174.5; HRMS
(MALDI–TOF) calcd for C132H222N40NaO35 [M+Na]+ 2950.6719,
observed 2950.6423.
Table 2
Synthesis of 13 and 14a–e
Product
mg 9
mg 1
mg 10
Yield,
MALDI-TOF
(equiv)
(equiv)
mg (%)
13
10
56
56
18
7.0
8.0
0
39 (16)
97 (45)a
63 (16)
44 (10)
17 (10)
20 (10)
4.5 (15)
13 (26)
3.0 (5)
44 (61)
14 (42)
22 (49)
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
Figure S6
14a
14b
14c
14d
14e
15 (0.34)
15 (0.34)
29 (2)
17 (3)
31 (5)
2.1.4.2. Procedure for reaction of azide-functionalized MSH4
derivative 1 and 1-azidohexane with 9 to produce multivalent
constructs 14a.
A mixture of 9 (56 mg, 56
mol), and tetrakis(acetoni-
l
mol), azide 118
a
1-Azidohexane used in place of 10.
(15 mg, 19 mol), TBTA (2.0 mg, 3.8
l
l

6478
V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
Figure 3. MALDI-TOF mass spectrum of 14e showing the distribution of products. The numbers indicate the number of attached MSH4 ligands. The insert shows the
anticipated statistical product distribution assuming attachment of 4.1 ligands per scaffold. For peak identification and for spectra of 13, 14a–14e, and 15a see the
Supplementary Data.
2.1.4.6. Procedure for reaction of azide-functionalized CCK4
derivative 11 and azide-functionalized serine amide derivative
10 with 9 to produce multivalent constructs 15b–d. A mixture
of 9 (variable amounts, see Table 3), azide 11 (variable amounts,
14c–14e as white powders; yields are given in Table 2. Products
were analyzed by MALDI-TOF (see Fig. 3 and Figs. S4–S6 in the
Supplementary data) and by UV spectroscopy.18
see Table 3), TBTA (0.43 mg/
trile)copper(I) hexafluorophosphate (0.3 mg/
(1.5 mL) was irradiated for 2 h in a Biotage microwave reactor
(100 °C). Azide 1018 (variable amounts, see Table 3) was then
added to the reaction mixtures and irradiation was resumed for
another 4 h. After the reactions were complete, water (2.5 mL/
l
mol of 9), and tetrakis(acetoni-
2.1.4.5. Procedure for reaction of azide-functionalized CCK4
derivative 11 and azide-functionalized serine amide derivative
10 with 9 to produce multivalent constructs 15a. A mixture of
lmol of 9) in dry DMF
9 (205 mg, 209
8.2 mol), and tetrakis(acetonitrile)copper(I) hexafluorophosphate
(3 mg, 8.2 mol) in dry DMF (1.5 mL) was irradiated for 2 h in a
lmol), azide 11 (30 mg, 41 lmol), TBTA (4.3 mg,
l
l
lmol of 9) was added and the mixtures were extracted with CHCl3
Biotage microwave reactor (100 °C). After the reaction was com-
plete, the DMF was evaporated under reduced pressure to afford a
residue that was subjected to silica gel chromatography (63–
containing dithizone (20 mg/L, 2 ꢀ 15 mL) to remove copper.35 The
water layers were then washed with DCM (2 ꢀ 15 mL) to remove
any remaining 10 and TBTA. After lyophilization, the residues were
fractionated by preparative HPLC (10?90% acetonitrile in water
containing 0.1% TFA within 50 min, tR 22–25 min) to afford
products 15b–d as white solids; yields are given in Table 3.
Products were analyzed by UV spectroscopy.18
210 lm) using DCM/methanol (9:1). After evaporation of the
volatile materials, the residue (40 mg) was taken up in dry DMF
(1.5 mL), azide 1018 (57 mg, 230
tetrakis(acetonitrile)copper(I)
l
mol), TBTA (8 mg, 16
hexafluorophosphate
l
mol), and
(6 mg,
16 lmol) were added, and the reaction mixture was irradiated
for 6 h in a Biotage microwave reactor (100 °C). Following workup
as described above for compounds 14c–e and lyophilization, the
residue was fractionated by preparative HPLC (10?90% acetoni-
trile in water containing 0.1% TFA within 50 min, tR 29 min) to
2.2. Biological studies
2.2.1. Formulation of solutions
Solutions of MSH4 and CCK4 constructs for binding assays were
made up in water and dimethylsulfoxide (HYBRI-MAX), respec-
tively, based on the expected incorporations of MSH4 or CCK4.
Concentrations of ligand in solution were then determined by mea-
surement of the UV absorbance at 280 nm using a standard calibra-
tion plot.18
afford 15a as white solid; yield 14 mg (4.1 lmol, 17%). Product 15a
was analyzed by MALDI-TOF (see Fig. S7 in the Supplementary
data) and by UV spectroscopy.18
Table 3
Synthesis of 15a–d
2.2.2. Binding assays
Product
mg 9
mg 11
mg 10
Yield,
MALDI-TOF
Quantitative receptor-binding assays were carried out following
a previously described method.11 Hek293 cells engineered to ex-
press both CCK2R and MC4R were used to assess ligand binding.
Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum. For MC4R assays, cells
were seeded in PerkinElmer, tissue culture treated, black-frame
with white well, 96-well plates (part # 6005060) at a density of
1.5 ꢀ 104 cells per well and were allowed to reach 80–90% conflu-
ence. For CCK2R assays, 2.5 ꢀ 105 cells were plated in each well of
(equiv)
(equiv)
mg (%)
15a
15b
15c
15d
205
17
19
30 (0.2)
25 (2)
41 (3)
40 (5)
57 (10)a
50 (12)
47 (10)
27 (10)
14 (17)a
15 (23)
11 (13)
15 (26)
Figure S7
nrb
nrb
11
nrb
a
Equivalents and yield based on 40 mg (23
intermediate; see Experimental Section for details.
MALDI-TOF spectra showed no ions that could be identified as belonging to
product. Yield determined by sample weight and UV spectroscopy.
lmol) of purified mono-CCK4
b

V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
6479
prepared by solid phase synthesis36,37 on Rink amide Tentagel S re-
sin as depicted in Scheme 3. 6-Azidohexanoic acid34 was coupled
to the N-terminus of the resin-bound tetrapeptide. Simultaneous
side chain deprotection and cleavage of the tetrapeptide from the
resin was effected using a mixture of trifluoroacetic acid, triisopro-
pylsilane, thioanisole, and water (91:3:3:3), producing the desired
azide-terminated ligand, N3(CH2)5(C@O)-Trp-Met-Asp-Phe-NH2
(11). The triazole-containing ligand CH3(CH2)3(C2N3)(CH2)5(C@O)-
Trp-Met-Asp-Phe-NH2 (12) was prepared by N-terminal acylation
of the resin-bound tetrapeptide with 6-(4-butyl-1H-1,2,3-triazol-
1-yl)hexanoic acid18 in place of 6-azidohexanoic acid. Compounds
11 and 12 were purified by reversed phase C18 preparative HPLC
(yields ranged from 39% to 46%) and were characterized by analyt-
ical HPLC and ESI-MS. Details appear in Table 1.
Table 4
Competitive binding of MSH4, CCK4, 12, 13, 14a–14e, 15a–15d, and 17 to MC4R or
CCK2R
Compound
MC4R
CCK2R
Kia (nM)
Kia
(lM)
nb
nb
MSH4
17
1.3 0.38
1.9 0.14
7.3 1.1
1.6 0.16
0.54 0.04
0.23 0.02
0.17 0.02
nd
5
5
4
4
4
4
4
ndc
nd
nd
nd
nd
nd
14a
14b
14c
14d
14e
CCK4
12
15a
15b
15c
15d
13
nd
3.1d
nd
nd
nd
nd
18 5.7
67 9.4
1.5 0.7
2.0 0.3
0.80 0.2
nbe
3
3
3
3
3
2
Reaction of 9 with an excess of 1018 in the presence of tetra-
kis(acetonitrile)copper(I) hexafluorophosphate and tris[(1-benzyl-
1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) in methanol in a Bio-
tage microwave reactor at 100 °C gave the corresponding serine
amide octamer 13 (Scheme 4). Copper ions were removed from
this mixture by complexation with dithizone and removal of the
complex by extraction with CHCl3.35 Other small organic-soluble
molecules (TBTA, excess 10) were also removed during this extrac-
tion. The water-soluble product 13 was purified by preparative re-
versed phase HPLC, recovered by lyophilization, and characterized
by MALDI-TOF mass spectrometry (see Fig. S1 in the Supplemen-
tary data).
nd
nbe
3
a
Ki values were calculated using the equation Ki = EC50/(1 + ([ligand]/KD)) where
[ligand] = 10 nM and KD = 8.3 nM for probe 4 and [ligand] = 2 nM and KD = 34.6 nM
for probe 16.
b
The value given represents the average of n independent competition binding
experiments, each done in quadruplicate.
c
Not determined.
d
e
Taken from Ref.30
.
Compound 13 was unable to inhibit the binding of probe 4 or probe 16 in the
concentration range tested (10ꢂ5–10ꢂ12 M in serine amide).
Reaction of 9 with varying amounts of 1 in the presence of cop-
per(I) and TBTA in methanol in a Biotage microwave reactor at
100 °C, followed by reaction either with an excess of 1-azidohex-
ane or with the serinamide-derived azide 10 under the same con-
ditions, gave the corresponding multimeric mixtures 14a–14e.
Copper ions and small organic molecules were removed as previ-
ously described. The crude product mixtures were further purified
by preparative reversed phase HPLC and were characterized by
MALDI-TOF mass spectrometry (see Fig. 3 and Figs. S2–S6 in the
Supplementary data). The average numbers of R(C@O)-MSH4-
NH2 ligands per scaffold were determined to be 1.0, 1.0, 1.3, 2.6,
and 4.1 for the multimeric mixtures 14a–14e, respectively, by UV
spectroscopy (see Section 4 and the Supplementary Data for
details).18
SigmaScreen poly-o-lysine coated 96-well, black/clear bottom
plates (catalog # M5307-SEA) and incubated for 48 h at 37 °C. On
the day of the experiment, media was removed from all wells. Li-
gands were diluted in binding buffer (MEM, 25 mM Hepes [pH
7.4], 0.3% BSA, 1 mM 1,10-phenanthroline, 0.5 mg/L leupeptin,
and 200 mg/L bacitracin). Test compound solutions (50
lL), in a
range of dilution concentrations, were added to 50 L of a 10 nM
l
solution of probe 4 (MC4R assay) or a 2 nM solution of probe 16
(CCK2R assay). Each concentration was tested in quadruplicate.
Cells were incubated in the presence of unlabeled and labeled li-
gands at 37 °C and 5% CO2 for 1 h. Following incubation, media
was removed and the wells washed 3ꢀ with wash buffer (50 mM
tris(hydroxymethyl)aminomethane hydrochloride, 0.2% BSA,
30 mM NaCl). Enhancement solution (PerkinElmer 1244-105)
Similarly, reaction of 9 with varying amounts of azide 11 in the
presence of copper(I) and TBTA in DMF in a Biotage microwave
reactor at 100 °C, followed by reaction with an excess of azide 10
under the same conditions, gave the corresponding multimeric
was added (100 lL/well) and plates were incubated for 30 min at
37 °C before measuring fluorescence using a VICTOR™ X4 2030
Multilabel Reader (PerkinElmer) instrument and the standard Eu
time-resolved fluorescence (TRF) measurement settings (340 nm
a,b
excitation, 400
615 nm).
ls delay, and emission collection for 400 ls at
FmocNH
H-Trp(Boc)-Met-Asp(t-Bu)-Phe-NH
Rink resin
Competitive binding data were analyzed with GraphPad Prism
software using nonlinear regression analysis and fitted to a classic
one site binding competition equation. Each EC50 value was gener-
ated from individual competitive binding assays and converted to a
Ki value using the equation Ki = EC50/(1 + ([ligand]/KD)) where [li-
gand] refers to the concentration of the probe used as the labeled
competed ligand. For probe 4, [ligand] = 10 nM and KD = 8.3 nM.
For probe 16, [ligand] = 2 nM and KD = 34.6 nM. Results are given
in Table 4. The value given represents the average of n independent
competition binding experiments.
c
N3 (CH2)5-C(=O)Trp(Boc)-Met-Asp(t-Bu)-Phe-NH
d
N3 (CH2)5-C(=O)Trp-Met-Asp-Phe-NH2
11
3. Results
C4H9
N (CH2)5-C(=O)Trp-Met-Asp-Phe-NH2
N
3.1. Chemistry
N
12
Reaction of sucrose with 24 equiv of sodium hydride in DMF,
followed by addition of 12 equiv of 6-bromo-1-hexyne33 afforded
octaalkyne 9 in 43% yield after chromatography (Scheme 2). Azides
1 and 10 were prepared as previously described.18 Azide 11 was
Scheme 3. Solid phase synthesis. Reagents: (a) piperidine; (b) Fmoc/tBu solid phase
synthesis; (c) N3(CH2)5CO2H, Cl-HOBt, DIC; (d) trifluoroacetic acid/triisopropylsi-
lane/thioanisole/water (91/3/3/3).

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V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
Scheme 4. Synthesis of multivalent compounds 13, 14a–14e, and 15a–d via CuAAC. The positions of ligand attachment are presumed to be random.
mixtures 15a–15d. Copper ions and small organic molecules
were removed as previously described. The crude product mixtures
were further purified by preparative reversed phase HPLC and
were characterized, in the case of 15a, by MALDI-TOF mass spec-
trometry (see Fig. S7 in the Supplementary Data). Satisfactory mass
spectra of 15b–15d could not be obtained. The average numbers of
R(C@O)-CCK4-NH2 ligands per scaffold were determined to be 1.0,
1.3, 2.2, and 3.6 for the multimeric mixtures 15a–15d, respectively,
by UV spectroscopy.18
4. Discussion
The one-step synthesis of octaalkyne 9 starting from sucrose
was reasonably efficient and can provide access to multigram
quantities of this product. Presumably, the identities of the sugar
starting material and the alkylating agent can be varied to produce
many such scaffolds. Microwave-driven CuAAC was used to attach
zero, one, or an average of 1.3, 2.6, or 4.1 copies of the MSH4 azide
1 to scaffold 9. The remaining alkyne residues of the scaffold were
then reacted with 1-azidohexane to produce 14a, or with serina-
mide-derived azide 10 to produce 13 and 14b–e. The constructs
so produced were purified from copper and from small molecules
by extraction, further purified by preparative reversed phase HPLC,
and characterized by MALDI-TOF mass spectrometry and by UV
spectroscopy. While MALDI-TOF analysis was reasonably consis-
tent with statistical attachment of ligands in the case of 14c, the
mass spectra for 14d and 14e suggested that ligand attachment
was not statistical (see Fig. 3 and Figs. S4–S6 in the Supplementary
data). MALDI-TOF analysis of a 1:1 mixture of monovalent and
divalent MSH4 constructs on a squalene scaffold17,27 demonstrated
a highly attenuated sensitivity for detection of the divalent con-
struct. Thus, MALDI-TOF analysis of multimers 14c–14e is qualita-
tive, with actual distributions of MSH4 ligands somewhere
between the MALDI-TOF limit of analysis and the expected statis-
tical distributions. For this reason, concentrations of multivalent
species for bioassays were determined by UV analysis (see calcula-
tions in the Supplementary data).
3.2. Bioassays
Hek293 cells overexpressing both MC4R22,38 and CCK2R30,39
were used to assess ligand binding using previously described
europium-based competitive binding assays that employed
Eu-DTPA-NDP-a-MSH-NH2 (4) or Eu-DTPA-CCK8-NH2 (16) as the
labeled probe.11 The Ki values for the parental ligands
CH3C(C@O)-His-DPhe-Arg-Trp-NH2 (listed as MSH4) and Trp-
Met-Asp-Phe-NH2 (listed as CCK4), for the corresponding tria-
zole-containing control compounds 12 and 1718, and for the con-
structs 13, 14a–14e, and 15a–15d are listed in Table 4.
Constructs 15a–15d bearing one or an average of 1.3, 2.2, or 3.6
CCK4 ligands were similarly prepared from scaffold 9, CCK4 azide
11, and azide 10. In principle, other ligands, imaging agents, and/
or therapeutic agents might be attached to scaffold 9.

V. Rao et al. / Bioorg. Med. Chem. 19 (2011) 6474–6482
6481
Constructs 13, 14a–e, and 15a–d were subjected to biological
testing using previously described competitive binding assays.11
Serinamide derivative 13 was ineffective at blocking probes 4
and 16 from binding to cells displaying MC4R and CCK2R over
the range of concentrations tested (Table 4). The Ki for the mono-
valent MSH4 control compound 17 was 1.5 times the value for
the parental ligand, indicating that attachment of the triazole-con-
taining ‘spacer’ to the N-terminus of MSH4 has a modest detrimen-
tal effect on ligand binding to MC4R.18 The Ki values differed
among the various sucrose-derived constructs 14a–e. Comparison
of the Ki values for 14a and 14b suggests that termination of the
non-MSH4-bearing sidechains with the more hydrophilic serina-
mide residues, as opposed to hydrophobic alkyl groups, enhances
construct binding. That this enhancement is not due to specific
binding by the serinamide residues is supported by the inactivity
of compound 13. While the Ki values decrease with higher levels
of MSH4 incorporation (compare 14b–e), the observed changes
can be attributed to statistics and proximity effects and suggest
effective, but monovalent binding of 14b–e at available MC4R.
These results are consistent with results from solanesol-derived
multivalent MSH4 constructs18 and divalent MSH4 constructs de-
rived from a flexible linker.8 One might suppose that 14b–e are
competent binders as monovalent species, but are incompetent
binders as multivalent species due to improper ligand spacing
and/or presentation for binding.40 However, as to ligand spacing,
estimates of the distance between ligand binding sites for adjacent
receptors range from 20 to 50 Å.9 If a spherical distribution of li-
gands is assumed for these sucrose-derived multivalent constructs,
the maximum distance between ligands is approximately 40 Å.41 If
a linear array of ligands is assumed for the solanesol-derived mul-
tivalent constructs, the distances between ligands are variable with
a maximum separation of approximately 90 Å.42 As to ligand pre-
sentation, it seems unlikely that all of the MSH4 multivalent and
bivalent constructs studied would improperly present a second li-
gand for binding, given the wide range of permitted N-terminal
and C-terminal modifications of this ligand43 and the fact that
the observed binding affinities for the first ligand are comparable
to the parental ligand.
Results from assays with CCK4 constructs 15a–d stand in con-
trast with results from assays with MSH4 constructs 14b–e. The
Ki for the monovalent CCK4 control compound 12 was six times
the value for the parental ligand, indicating that attachment of
the triazole-containing ‘spacer’ to the N-terminus of CCK4 has a
significant detrimental effect on ligand binding to CCK2R. The Ki
values differed among the various sucrose-derived constructs
15a–d. The Ki value for 15a which bears one copy of the CCK ligand
was 22 times the value for the parental ligand and 3.7 times the va-
lue for the control compound 12. Apparently, attachment of the
CCK4 ligand to the sucrose-derived scaffold has a much greater
negative effect on binding than was observed for MSH4. This
observation is in keeping with the more restrictive limits on N-ter-
minal substitution for this ligand.46 The Ki values decrease with
higher levels of CCK4 incorporation, with 15b–d of comparable po-
tency and each 30–80 times as potent as 15a, suggestive of multi-
valent binding. Since the affinity of the parental CCK4 ligand for its
receptor is 420 times that of the parental MSH4 ligand for its
receptor, it seems reasonable to assume that the off-rates of
15a–d are lower than the off-rates for 14b–e, perhaps providing
sufficient time for a second binding event to occur before dissoci-
ation of the monovalently bound construct. The fact that the great-
er multivalency of 15d does not more considerably enhance the
avidity observed for 15c or 15b may be a consequence of the spa-
tial distribution of the CCK4 ligands attached to this spherical scaf-
fold, which necessitates that on average half of the ligands must be
oriented away from the cell surface. This fact limits the number of
simultaneous attachments that can be made through ligand bind-
ing. For a fully loaded sucrose-derived scaffold bearing eight copies
of ligand, there can be at most four simultaneous attachments. For
steric and/or electrostatic reasons, it is unlikely that full scaffold
loading with ligand could commonly be achieved. Assuming half
loading (four ligands per scaffold) or less, as is the case for 14c–e
and 15b–d, the analysis above suggests that bivalent attachment
is a more realistic expectation.
Acknowledgments
This work was supported by grants R33 CA 95944, RO1 CA
97360, RO1 CA 123547, and P30 CA 23074 from the National Can-
cer Institute.
CH3(CH2)3
(CH2)5(CO)-His-
DPhe-Arg-Trp-NH2
N
N
N
17
Supplementary data
Monovalent binding of multivalent MSH4 constructs would be
expected if the off rates of monovalently bound constructs are fas-
ter than the binding of a second ligand arm to MC4R on the cell
Supplementary data (mass spectra of multivalent constructs 13,
14a–e, and 15a, analysis of ligand distribution for 14c–e and 15b–
d, and 1H and 13C NMR spectra of compounds 9 and 13) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.08.053.
surface. The NDP-a-MSH ligand, upon which 4 is based, is known
to have a slow off-rate (t1/2 ꢃ 8 h).44 The off rates of MSH4 and re-
lated constructs, such as 14a–b and 17, are unknown, but are ex-
pected to be much larger than the rates for NDP-a-MSH and
References and notes
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observations are consistent with fast off-rates for monovalently
bound MSH4 constructs.
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