Squalene-derived flexible linkers for bioactive peptides

Article abstract of DOI:10.1016/j.bmcl.2007.04.001

A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-α-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3 + 2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-α-MSH constructs, presumably due to slow off rates for this high affinity ligand.

Full text of DOI:10.1016/j.bmcl.2007.04.001

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3310–3313
Squalene-derived flexible linkers for bioactive peptidesq
Bhumasamudram Jagadish,a Rajesh Sankaranarayanan,a Liping Xu,b Reyniak Richards,a
Josef Vagner,a Victor J. Hruby,a,b Robert J. Gilliesb and Eugene A. Masha,*
aDepartment of Chemistry, University of Arizona, Tucson, AZ 85721-0041, USA
bDepartment of Biochemistry and Molecular Biophysics, The Arizona Cancer Center, Tucson, AZ 85724-5024, USA
Received 9 March 2007; accepted 2 April 2007
Available online 6 April 2007
Abstract—A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two
steps from squalene and was used to connect two high affinity NDP-a-MSH ligands or two low affinity MSH(4) ligands. The ligands
were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-cat-
alyzed ‘click’ 3 + 2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the
human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental
ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand
MSH(4), but not for dimeric NDP-a-MSH constructs, presumably due to slow off rates for this high affinity ligand.
Ó 2007 Elsevier Ltd. All rights reserved.
Early detection of many human cancers would be facil-
itated by the availability of reagents that could seek out
and selectively bind to cancer cells and report their exis-
tence and location by non-invasive molecular imaging.1
Our strategy for development of such reagents involves
linking reporter moieties to multivalent ligands that con-
tain multiple copies of individual binding units that
hence cooperatively bind to cell surface receptors that
are overexpressed in cancer cells.2 Multivalent molecules
should display enhanced affinity and selectivity for such
cells.2,3
ported multivalent binding3b,5 have prompted a reexam-
ination of this earlier approach with the intent of
developing a more soluble biocompatible polymer scaf-
fold and more efficient ligand attachment chemistry.
Herein we present model synthetic and in vitro biologi-
cal studies relevant to these goals.
The copper-catalyzed 3 + 2 cyclization of azide and
alkyne moieties to generate triazole products6 was an
obvious choice to replace the maleimide electrophile/
thiol nucleophile and thiol/disulfide redox attachment
chemistries used previously with PVA.4
The foundation for ligand-guided multivalent attach-
ment of reporter moieties to cell surfaces bearing tar-
geted receptors was laid in part by studies that
employed a poly(vinyl alcohol) (PVA) scaffold deco-
rated with fluorescein and NDP-a-MSH ligands.4 Such
molecules bound specifically and irreversibly to mouse
and human melanoma cells that expressed and displayed
melanocortin receptors. The PVA-based system was not
extended to other peptide hormone/receptor systems due
to problems with the attachment chemistry and the
insolubility of PVA. Recent advances in polymer-sup-
The search for a PVA replacement was guided by the
observation that incompletely hydrolyzed PVA is often
more water-soluble at room temperature than is a more
completely hydrolyzed PVA.7 This is presumably due to
interruption of hydrogen bonded microcrystalline do-
mains, and suggested that a polymer bearing fewer, ste-
reorandom hydroxyl groups might exhibit less
crystallinity and greater water solubility. Such a polymer
can be prepared from polyisoprene.8 Since analysis of a
polymer product is complicated by high molecular
weight and polydispersity, we chose to employ squalene
as a more tractable model system for the establishment
of the synthetic methodology necessary for this
approach.
Keywords: Multimeric; Ligands; Click chemistry; Melanocyte stimu-
lating hormone; NDP-a-MSH.
q
This paper is dedicated to Professor C. Dale Poulter on the occasion
of his 65th birthday.
Corresponding author. Tel.: +1 520 621 6321; fax: +1 520 621
8407; e-mail: emash@u.arizona.edu
Hydroboration and oxidation of squalene9 produced a
mixture of hexaols 1 (Scheme 1).10 The crude product
*
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.001

B. Jagadish et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3310–3313
3311
OH
OH
OH
CH3
CH3
CH3
CH3
squalene
H3C
a
CH3
CH3
CH3
O
OH
OH
N
OH
OH
OH
CH3
CH3
CH3
CH3
N
N
O
X
9
H3C
CH3
CH3
CH3
OH
OH
OH
X
1
O
N
N
N
b
O
OH
OH
CH3
CH3
CH3
OH
OH
OH
CH3
CH3
CH3
CH3
H3C
CH3
H3C
CH3
CH3
CH3
O
OH
OH
CH3
CH3
CH3
O
OH
N3
OH
N
N
N
O
X
10
3
N3
Compound
X
O
OH
OH
CH3
CH3
CH3
CH3
9a
10a
9b
10b
9c
Ser-NH2
Ser-NH2
NDP-α-MSH-NH2
NDP-α-MSH-NH2
MSH(4)-NH2
H3C
CH3
CH3
CH3
O
OH
N3
OH
4
Scheme 1. Reagents: (a) BH3, THF, H2O2, NaOH; (b) NaH, DMF,
Br(CH2)6N3 (2). The sites of attachment of the 6-azidohexyl moieties
to the squalane scaffold shown are arbitrary. Mixtures of all possible
stereoisomers of 1 and regioisomers of 3 and 4 are produced.
10c
MSH(4)-NH2
Figure 1. Monovalent and divalent compounds on a squalene-derived
scaffold prepared via ‘click’ attachment of alkynylated ligands. The
sites of ligand attachment shown are arbitrary.
0.17 mmol/g).16 The product resin retained all side-chain
protecting groups. NHS ester 6 was coupled to the
N-terminus of the resin-bound peptide. Simultaneous
side chain deprotection and cleavage of the peptide from
the resin was effected using a mixture of trifluoroacetic
acid, 1,2-ethanedithiol, thioanisole, and water (91/3/3/
3), producing the desired alkynylated NDP-a-MSH
ligand 11. An alkynylated derivative, 12, of the low
affinity ligand MSH(4)17 was similarly prepared. Com-
pounds 11 and 12 were purified by reverse-phase C18
preparative HPLC and were characterized by ESI-MS
and MALDI-TOF. Details appear in Table 1.
was purified by column chromatography (67% yield)
and analyzed by NMR and HRMS. These analyses con-
firmed that secondary alcohols were highly predominant
and that a mixture of diastereomers had been pro-
duced.11 Reaction of 1 with 2.2 equivalents of sodium
hydride in DMF, followed by addition of 4.0 equiva-
lents of 1-azido-8-bromohexane (2),12 afforded, after
chromatography, monoazides 3 and bisazides 4 in 38%
and 24% yields, respectively, as mixtures of regioisomers.
Trace amounts of trisazides and higher homologs were
also obtained.
To demonstrate the feasibility of click attachment to 3
and 4, alkyne 7 was prepared from serine amide hydro-
chloride (5)13 and 2,5-dioxopyrrolidin-1-yl hex-5-ynoate
(6, Scheme 2). Reaction of 3 and 4 with 7 in the presence
of the catalyst derived from tetrakis(acetonitrile)cop-
per(1) hexafluorophosphate and tris-(benzyltriazolylm-
ethyl)-amine (TBTA, 8)14 in t-BuOH/water 2:1 gave
the corresponding triazoles 9a and 10a (Fig. 1) in 84%
and 80% yields, respectively, after chromatography.
O
Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val NH2
11
O
His-DPhe-Arg-Trp NH2
12
The high affinity ligand NDP-a-MSH15 was constructed
on Rink amide Tentagel S resin (initial loading
Reaction of azides 3 and 4 with 11 in methanol in the
presence of the copper/TBTA catalyst gave the corre-
HO
H2N
HO
O
O
O
a
H2N
N
N
NH2•HCl
O
H
O
O
O
5
6
7
Scheme 2. Reagents: (a) Et3N, DMF, H2O.

3312
B. Jagadish et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3310–3313
Table 1. Mass spectral and HPLC characterization of compounds 9b and c, 10b and c, 11, and 12
a
tR
Compound
Formula
Calcd mass [Ion]
Mass found (error)
K0
9b
C118H188N24O25
C206H314N48O44
C74H120N14O11
C118H178N28O16
C82H115N21O19
781.4804 [M+3]3+
1042.1030 [M+4]4+
691.4709 [M+2]2+
748.8070 [M+3]3+
566.9638 [M+3]3+
738.3840 [M+1]1+
781.4833 (3.7 ppm)
1042.1055 (2.4 ppm)
691.4693 (2.4 ppm)
748.8052 (2.4 ppm)
566.9653 (2.6 ppm)
738.3829 (1.4 ppm)
35.3–36.4
33.4–34.7
34.8–37.3
32.9–36.9
20.41
nab
nab
nab
nab
8.66
6.02
10b
9c
10c
11
12
C
38H47N11O5
15.48
a Linear gradient of from 10% to 60% CH3CN in 0.1% aqueous TFA over 50 min.
b Not applicable, this compound is a regiochemical and stereochemical mixture.
sponding triazoles 9b and 10b, while reaction of 3 and 4
with 12 gave the corresponding triazoles 9c and 10c
(Fig. 1). These compounds were purified by preparative
reverse-phase C18 HPLC and were characterized by ana-
lytical HPLC and by ESI-MS and MALDI-TOF. De-
tails appear in Table 1.
In contrast, the IC50 values for compounds 9c [scaf-
fold + MSH(4)] and 10c [MSH(4) + scaffold + MSH(4)]
were higher and lower, respectively, than the values
for the alkynylated MSH(4) control compound 12 and
the parental MSH(4) ligand, which were similar. These
results indicate that acylation of the N-terminus of
MSH(4) had little or no effect on the binding to
hMC4R, that attachment of MSH(4) to the squalene-de-
rived scaffold had a modest detrimental effect on the
binding, and that statistical and proximity effects re-
sulted in the lowering of the IC50 for 10c relative to 9c
by an order of magnitude.
HEK293 cells overexpressing the human melanocortin 4
receptor (hMC4R) were used to assess ligand binding,18
which was evaluated using a previously described lan-
thanide (Eu) based competitive binding assay.19,20 Table
2 lists the IC50 values (averaged over n experiments) for
the serine amide-containing compounds 7, 9a, and 10a,
the ligand NDP-a-MSH and the NDP-a-MSH-contain-
ing compounds 11, 9b, and 10b, and the ligand MSH(4)
and the MSH(4)-containing compounds 12, 9c, and 10c.
This model study has demonstrated that a poly(1,5-diol)
can be prepared from a polyisoprene (squalene) and
modified by alkylation with a 1-azido-x-bromoalkane
to introduce terminal azide moieties; that functionally
protected peptides on a solid support can be N-termi-
nally acylated with active x-alkynyl esters to introduce
alkyne moieties that survive deprotection and cleavage
from the resin; that the above azides and alkynes can
be joined by copper-catalyzed 3 + 2 cyclization to form
triazoles that can be purified and characterized; and that
attachment in this manner of NDP-a-MSH and MSH(4)
ligands to the polyisoprene-derived scaffold does not sig-
nificantly interfere with ligand binding to the hMC4
receptor at the cell surface. Extension of this work to
polymeric polyisoprene-derived scaffolds and to other li-
gand/receptor combinations is underway.
As expected, serine amide derivatives 7, 9a, and 10a
were ineffective at displacing Eu-NDP-a-MSH over the
range of concentrations tested (10ꢀ5–10ꢀ12 M).
The IC50 values for compounds 9b [scaffold + NDP-a-
MSH], 10b [NDP-a-MSH + scaffold + NDP-a-MSH],
and the alkynylated NDP-a-MSH control compound
11 were all somewhat lower than the value for the paren-
tal NDP-a-MSH ligand. These results indicate that acyl-
ation of the N-terminus of NDP-a-MSH modestly
enhanced binding to hMC4R, and that attachment of
NDP-a-MSH to the squalene-derived scaffold had no ef-
fect on the binding. No statistical halving of the IC50
was observed for 10b relative to 9b. This is presumably
due to slow release of the ligand from the receptor, as
previously described for short, rigid linkers.21
Acknowledgments
TBTA was a generous gift from Professor K.B. Sharp-
less of The Scripps Research Institute. This work was
supported by Grants R33 CA 95944, RO1 CA 97360,
and P30 CA 23074 from the National Cancer Institute.
Table 2. Competitive binding of NDP-a-MSH, MSH(4), 7, 9a–c,
10a–c, 11, and 12 to hMC4R
Compound
IC50
na
7
9a
nab
3
3
3
5
4
5
6
4
4
7
7
nab
Supplementary data
10a
nab
NDP-a-MSH
11
9b
5.9 1.9 nM
3.2 0.9 nM
3.9 1.2 nM
3.3 0.8 nM
1.1 0.5 lM
0.9 0.1 lM
3.3 0.5 lM
0.4 0.2 lM
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.04.001.
10b
MSH(4)
12
9c
10c
References and notes
a The IC50 value given is the average of n independent binding exper-
iments, each done in quadruplicate.
b This compound was unable to displace Eu-NDP-a-MSH in the
concentration range tested (10ꢀ5–10ꢀ12 M).
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Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% FBS. Cells were plated in Black and
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), 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 excitation, 400 ls delay, and emission collection
for 400 ls at 615 nm). Competition curves were analyzed
with GraphPad Prism Software using the sigmoidal dose–
response classical equation for non-linear regression
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