Design, synthesis, and validation of rigid linkers for bioactive peptides

Article abstract of DOI:10.1016/j.tetlet.2005.08.151

Rigid linkers of variable length were synthesized and used to connect two NDP-α-MSH ligands. The linkers were incorporated by solid-phase synthesis. Biological evaluations indicate that there is virtually no effect of these linkers on ligand binding to th

Full text of DOI:10.1016/j.tetlet.2005.08.151

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 7589–7592
Design, synthesis, and validation of rigid linkers for
bioactive peptides
Yasunari Monguchi,a Josef Vagner,a Heather L. Handl,b Umasish Jana,a
Lucinda J. Begay,a Victor J. Hruby,a,b Robert J. Gilliesb and Eugene A. Masha,*
aDepartment of Chemistry, The University of Arizona, Tucson, AZ 85721-0041, USA
bDepartment of Biochemistry and Molecular Biophysics, The Arizona Cancer Center, Tucson, AZ 85724-5024, USA
Received 28 June 2005; revised 23 August 2005; accepted 25 August 2005
Available online 13 September 2005
Abstract—Rigid linkers of variable length were synthesized and used to connect two NDP-a-MSH ligands. The linkers were incor-
porated by solid-phase synthesis. Biological evaluations indicate that there is virtually no effect of these linkers on ligand binding to
the human melanocortin 4 receptor.
Ó 2005 Elsevier Ltd. All rights reserved.
In support of our effort to develop new strategies for
early detection and treatment of cancers,1 we require
linkers for tethering two or more peptide ligands. The
resulting multivalent molecules could display enhanced
affinity for targeted cell surface receptors.2 The distance
that must be spanned between targeted receptors on the
cell surface will vary, depending on the number, kind,
and mobility of the receptors, but is expected to exceed
the typical dimensions of small molecules. Several small
molecule linkers connected in series will, therefore, be
necessary to span the distance between receptors. For
peptide hormones, linkers should terminate in amine
and carboxylic acid functional groups to facilitate their
incorporation by solid-phase peptide synthesis. Both
flexible linkers3 and rigid linkers4 are of interest.5 Each
new linker must be validated as non-interfering with
the binding of the ligand. We report herein the syntheses
of suitably protected derivatives of rigid linkers 1–4,
which offer variable length, attachment of one or two
a-melanocyte stimulating hormone (a-MSH) peptide
ligands to these linkers by solid-phase synthesis, and
preliminary evaluations of the bioactivities of these
linker-modified peptides.
CO2H
CO2H
CO2H
CO2H
NH2
NH2
NH2
NH2
1
2
3
4
to derivatives of compound 2 has been described by
Manku et al.7 Accordingly, amino acid 2 was prepared
from p-aminomethylphenylboronic acid hydrochloride
(5)8 and methyl 4-bromobenzoate (7)9 and was con-
verted to the N-trifluoroacetamide derivative 9 in 74%
yield by treatment with trifluoroacetic anhydride in pyr-
idine (Scheme 1). Linkers 2–4 were incorporated as the
corresponding N-TFA derivatives to enhance solubility
under the conditions of solid-phase synthesis.
The commercially available6 N-Fmoc derivative of 1 was
incorporated into peptide structures using solid-phase
synthesis (vide infra). An efficient synthesis leading
New amino acids 400-aminomethyl-[1,10:40,100-terphenyl]-
4-carboxylic acid 3 and 6-(4-aminomethylphenyl)-2-
naphthalenecarboxylic acid 4 further increase the dis-
tance spanned by the linker.10 The synthesis of the N-
TFA derivative 13 of terphenyl linker 3 is depicted in
Scheme 2. Suzuki–Miyaura cross-coupling of 6 with
*
Corresponding author. Tel.: +1 520 621 6321; fax: +1 520 621
8407; e-mail: emash@u.arizona.edu
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.08.151

7590
Y. Monguchi et al. / Tetrahedron Letters 46 (2005) 7589–7592
CO2Me
B(OH)2
CO2Me
CO2Me
B(OH)2
B(OH)2
Br
NHCOCF3
CF3CO2Et
7
6
Et3N
MeOH
83%
Pd(PPh3)4 (cat)
Pd(PPh3)4 (cat)
Br
KF
KF
THF
reflux
100%
NHCOCF3
NH2·HCl
THF
reflux
87%
5
6
14
NHCOCF3
15
CO2Me
CO2H
CO2H
CO2H
CO2H
(CF3CO)2O
NaOH
THF
MeOH
97%
pyridine
74%
(CF3CO)2O
NaOH
NHCOCF3
NH2
NHCOCF3
THF
MeOH
100%
pyridine
96%
9
2
8
Scheme 1. Synthesis of the N-TFA derivative 9 of 40-aminomethyl-
biphenyl-4-carboxylic acid (2).
NH2
NHCOCF3
4
16
Scheme 3. Synthesis of the N-TFA derivative 16 of 6-(4-aminometh-
ylphenyl)-2-naphthalenecarboxylic acid (4).
B(OH)2
CHO
CO2Me
duced the corresponding amino acid 3 in quantitative
yield. The trifluoroacetamide moiety was reintroduced
to afford the target amino acid derivative 13.
MnO2
NaCN
NHCOCF3
6
The synthesis of the N-TFA derivative 16 of phenyl-
naphthyl linker 4 was synthesized in a similar manner
(Scheme 3). Cross-coupling of 6 with methyl 6-bromo-
2-naphthoate (14)12 afforded the N-protected amino
ester 15 in excellent yield. Hydrolysis of 15 using NaOH
gave the amino acid 4. Subsequent TFA-protection of
the amino group produced the target amino acid 16.
MeOH
87%
Pd(PPh3)4 (cat)
KF
THF
reflux
71%
Br
Br
11
10
CO2Me
CO2H
CO2H
The synthesis of linked peptides 17–19 consisting of two
NDP-a-MSH ligands13 connected in a head-to-tail fash-
ion by linkers 1–3 is depicted in Scheme 4. Also shown
in Scheme 4 is the synthesis of 20–22, consisting of
one NDP-a-MSH ligand12 connected at the N-terminus
through the carboxylate of linkers 1–3.
(CF3CO)2O
NaOH
dioxane
THF
MeOH
100%
pyridine
82%
The tridecapeptide NDP-a-MSH was constructed on
PS-Rink resin (23, initial loading 0.17 mmol/g, 1%
DVB).14 Resin 24 retained all side chain protecting
groups. The N-protected linkers were coupled to the
N-terminus of 24, giving resins 25–27. The N-Fmoc
group of 25 was cleaved by treatment with piperidine
and the N-TFA groups of 26 and 27 were cleaved by
treatment with 15% hydrazine and 15% methanol in
THF.15
NHCOCF3
12
NH2
NHCOCF3
13
3
Scheme 2. Synthesis of the N-TFA derivative 13 of 400-aminomethyl-
[1,10:40,100-terphenyl]-4-carboxylic acid (3).
40-bromo-[1,10-biphenyl]-4-carboxylic acid methyl ester
11, which was prepared by oxidation of aldehyde 1011
in MeOH, using catalytic Pd(PPh3)4 and KF afforded
terphenyl 12. Saponification of 12 using NaOH pro-
The resulting resins 28–30 were each split into two por-
tions. For the synthesis of 17–19, the free amine groups
of resins 28–30 were coupled with Fmoc-valine and
solid-phase peptide synthesis13 continued to complete

Y. Monguchi et al. / Tetrahedron Letters 46 (2005) 7589–7592
7591
a,b
FmocNH
23
H-Ser(tBu)-Tyr(tBu)-Ser(tBu)-Nle-Glu(OtBu)-His(Trt)-D-Phe-Arg(Pbf)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val
c
NDP-α-MSH
H
N
O
24
R
n
NDP-α-MSH
H
N
H
N
O
O
Ac
Ac
f
25 n = 1 R = Fmoc
26 n = 2 R = TFA
27 n = 3 R = TFA
n
n
NDP-α-MSH
NDP-α-MSH
37 n = 1
38 n = 2
39 n = 3
20 n = 1
21 n = 2
22 n = 3
a (25)
or
d (26–27)
e
H2N
O
NDP-α-MSH NH
O
b
e
n
NDP-α-MSH
n
NDP-α-MSH
31 n = 1
28 n = 1
29 n = 2
30 n = 3
32 n = 2
33 n = 3
Ac-NDP-α-MSH NH
O
Ac-NDP-α-MSH NH
O
f
n
n
NDP-α-MSH
NDP-α-MSH
34 n = 1
35 n = 2
36 n = 3
17 n = 1
18 n = 2
19 n = 3
Scheme 4. Solid-phase synthesis of NDP-a-MSH peptides 17–22. Reagents and conditions: (a) piperidine; (b) Fmoc/tBu solid-phase synthesis (Ref.
13); (c) N-protected linker, HOCt-DIC, DMF; (d) THF/MeOH/N2H4 (70/15/15); (e) pyridine/Ac2O (90/10); (f) CF3CO2H/HSCH2CH2SH/PhSMe/
H2O (91/3/3/3).
the second NDP-a-MSH sequence, giving resins 31–33.
The peptide was then capped by N-acetylation, giving res-
ins 34–36. Simultaneous side chain deprotection and
cleavage of the peptides from the Rink resin was effected
using a mixture of trifluoroacetic acid, 1,2-ethanedithiol,
thioanisole, and water (91/3/3/3) that produced the de-
sired compounds 17–19. For the synthesis of 20–22, resins
28–30 were terminally N-acetylated to give resins 37–39.
Treatment with the CF3CO2H/HSCH2CH2SH/PhSMe/
H2O cocktail effected simultaneous side chain deprotec-
tion and cleavage of the peptides from the resin and gave
compounds 20–22. Peptides 40 and 41 incorporating the
phenylnaphthyl linker 4 were similarly prepared.
Peptides 17–22, 40, and 41 were purified by reverse-
phase C18 preparative HPLC and were characterized
by ESI-MS and MALDI-TOF.15
Ligand binding was evaluated using a previously de-
scribed lanthanide based binding assay.16 HEK293 cells
overexpressing the human melanocortin 4 receptor
(hMC4R) were used to assess ligand binding.17 The
coding region of the hMC4R gene was expressed in
pcDNA3.1 (Invitrogen). HEK293/hMC4R cells were
grown in DulbeccoÕs Modified EagleÕs Medium
(DMEM) supplemented with 10% FBS. Cells were pla-
ted in Black & White Isoplates (Wallac, 1450-583) at a
density of 12,000 cells/well and were allowed to grow
for 3 days. On the day of the experiment, media were
aspirated from all wells. Ligands of interest were diluted
in binding buffer (DMEM, 1 mM 1,10-phenanthroline,
200 mg/L Bacitracin, 0.5 mg/L Leupeptin, and 0.3%
BSA) to result in final dilutions ranging from 10 lM
to 4 pM. Eu-labeled NDP-a-MSH was used at a final
concentration of 10 nM. Fifty microliters of the ligand
of interest and 50 lL of Eu-NDP-a-MSH were added
to each well and plates were incubated for 40 min
at 37 °C. Following the incubation, cells were washed
four times with Wash Buffer (50 mM Tris–HCl, 0.2%
BSA, and 30 mM NaCl) using Molecular Devices
NDP-α-MSH
Ac-NDP-α-MSH NH
O
40
NDP-α-MSH
Ac NH
O
41

7592
Y. Monguchi et al. / Tetrahedron Letters 46 (2005) 7589–7592
18, and 40 effectively removes the tethered, unbound
ligands from the ligand pool. Work to confirm this
hypothesis is currently underway.
100
75
50
25
0
Acknowledgments
This work was supported by grants R33 CA 95944 and
RO1 CA 97360 from the National Cancer Institute.
-10
-9
-8
-7
-6
-5
Supplementary data
Log [Ligand], M
Details of the syntheses of compounds 2–4, 6, 8–13, 15,
Figure 1. Representative ligand binding curves generated through
competitive ligand binding analysis comparing the binding affinities of
ligands 18 (h) and 21 (j). The calculated IC50 for 18 is 8 nM with an
R2 value = 0.91 and for 21 10 nM with an R2 value = 0.93.
1
and 16 and H NMR and 13C NMR spectra of com-
pounds 2–4, 8, 9, 11–13, 15, and 16. Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2005.08.151.
SkanWasher. Enhancement solution (Perkin Elmer,
1244-105) was added (100 lL/well) and the plates were
incubated for 30 min at 37 °C prior to reading. The
plates were read on a Wallac VICTOR3 instrument
using the standard Eu TRF measurement (340 nm exci-
tation, 400 ls delay, and emission collection for 400 ls
at 615 nm). Competition curves (e.g., Fig. 1) were ana-
lyzed with GraphPad Prism Software using the sigmoi-
dal dose–response classical equation for non-linear
regression analysis. Each data point represents the aver-
age of four samples, with the error bars indicating stan-
dard error of the mean.
References and notes
1. Handl, H. L.; Vagner, J.; Han, H.; Mash, E.; Hruby, V. J.;
Gillies, R. J. Expert Opin. Ther. Targets 2004, 8, 565–
586.
2. (a) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew.
Chem., Int. Ed. 1998, 37, 2754–2794; (b) Kiessling, L. L.;
Gestwicki, J. E. Curr. Opin. Chem. Biol. 2000, 4, 696–
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3. (a) Vagner, J.; Handl, H. L.; Gillies, R. J.; Hruby, V. J.
Bioorg. Med. Chem. Lett. 2004, 14, 211–215; (b) Raju, N.;
Ranganathan, R. S.; Tweedle, M. F.; Swenson, R. E.
Tetrahedron Lett. 2005, 46, 1463–1465.
Table 1 lists the IC50 values (averaged over n experi-
ments) for the ligand NDP-a-MSH, as well as for pep-
tides 17–22, 40, and 41. Interestingly, the IC50 values
for 17, 18, and 40 were virtually the same as for NDP-
a-MSH and the controls 20, 21, and 41. These results
indicate that the phenyl, biphenyl, and phenylnaphthyl
linkers have little or no effect on ligand binding to
hMC4R. Interestingly, compounds 19 and 22 that incor-
porate the terphenyl linker showed modest increases in
IC50. In addition, the expected statistical halving of
the IC50 for compounds containing two NDP-a-MSH
ligands was observed only for 19 and 22. This is consis-
tent with the observation that these compounds bind
less well to hMC4R. Tight binding in the cases of 17,
4. Maison, W.; Frangioni, J. V.; Pannier, N. Org. Lett. 2004,
6, 4567–4569.
5. On balance the series of linkers should be hydrophilic, but
short hydrophobic segments may be tolerated, if not
beneficial.
6. Purchased from Torviq, Niles MI.
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379–391.
8. Purchased from CombiBlocks, San Diego, CA.
9. Purchased from Aldrich Chemical Company.
˚
10. Distances between the benzylic carbon atoms: 1, 5.8 A; 2,
˚
˚
˚
10.0 A; 3, 14.3 A; and 4, 12.2 A; determined using AM1 in
Spartan Õ02 v1.0.4e.
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M. H.; Heward, C. B.; Burnett, J. B.; Hadley, M. E. Proc.
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Gillies, R. J. Anal. Biochem. 2004, 330, 242–250.
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Gantz; see: Gantz, I.; Miwa, H.; Konda, Y.; Shimoto, Y.;
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Biol. Chem. 1993, 268, 15174–15179.
Table 1. Competitive binding of NDP-a-MSH and 17–22, 40, and 41
to hMC4R
Compound
IC50 (nM)
na
NDP-a-MSH
5.6
3.5
5.0
5.6
5.2
8.4
16.5
3.7
3.9
3
4
4
5
5
5
5
5
4
17
20
18
21
19
22
40
41
a The IC50 value given is the average of n independent binding experi-
ments, each done in quadruplicate.