Near-infrared fluorescent divalent RGD ligand for integrin αvβ3-targeted optical imaging

Article abstract of DOI:10.1016/j.bmcl.2012.07.044

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)]₂ (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α_vβ₃-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α_vβ₃ binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.

Full text of DOI:10.1016/j.bmcl.2012.07.044

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5405–5409
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Near-infrared fluorescent divalent RGD ligand for integrin
optical imaging
avb3-targeted
⇑
Yunpeng Ye, Walter Akers, Baogang Xu, Sharon Bloch, Charles Odonkor, Samuel Achilefu
Department of Radiology, Optical Radiology Laboratory, Washington University School of Medicine, 4525 Scott Avenue, Saint Louis, MO 63108, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)]2 (1),
Received 16 May 2012
Revised 10 July 2012
Accepted 12 July 2012
Available online 20 July 2012
was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for
integrin
a
vb3-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited
vb3 binding affinity and tumor uptake in nude mice of A549.
remarkable improvements in integrin
a
The results suggest that cypate-linked divalent ligands can serve as an important molecular platform
for exploring receptor-targeted optical imaging and treatment of various diseases.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Integrin avb3
RGD peptide
Divalent ligand
Optical imaging
Cypate
Among the family of 24 integrins, integrin
important target for cancer imaging and therapy. This is because
a
vb3 has become an
We previously reported a series of carbocyanine-based near-
infrared (NIR) fluorescent probes for tumor-targeted optical imag-
ing in vivo.12,13,15–21 Noteworthy, a dicarboxylic acid-containing
carbocyanine analog (cypate; Fig. 1) can be used for facile conjuga-
tions of various bioactive molecules on solid support and in solu-
tion for molecular targeting of proteins expressed on plasma
the up-regulation of integrin vb3 in cancer and endothelial cells
a
is implicated in the progression and metastasis of various tumors.
Significant progress has been made in the discovery and develop-
ment of integrin
avb3-specific lactam-based cyclic RGD peptide
analogs such as cilengitide and c(RGDfK) for cancer therapy, as well
as targeted delivery of cancer imaging and therapeutic agents.1–9
Nevertheless, it remains a great challenge to leverage such mole-
cules into successful clinical applications due to the complexities
of integrin signaling network in cancer cells, tumor microenviron-
ments, and in vivo environments. For example, Reynolds et al.10 re-
ported that nanomolar concentrations of RGD-mimetic integrin
membrane, including integrin avb3 and somatostatin receptors.
Cypate has also served as a robust scaffold for conveniently con-
structing novel divalent and multivalent fluorescent molecules
for tumor-targeted optical imaging. In the present work, we aimed
to synthesize and evaluate a novel integrin avb3-targeted divalent
RGD ligand based on cypate for potential use in optical imaging of
tumors. We hypothesized that 1 can simultaneously bind two adja-
avb3 and
avb5 inhibitors can promote VEGF-mediated tumor angi-
cent avb3 integrins to enhance the avb3-binding affinity and tu-
ogenesis and tumor growth in mice, which may seriously compro-
mor-targeting with improved imaging contrast compared with its
mise the therapeutic efficacy of this class of compounds. Therefore,
monovalent counterpart 2.
novel approaches for exploiting integrin
a
vb3 targeting are still
Initially, we prepared RGD peptide-dye conjugates from cypate
and the non-protected cyclic peptide c(RGDfK) or c(RGDyK) via
the reaction between two N-hydroxysuccinimidyl (NHS) esters of
cypate and the amine group at the side chain of lysine residue as
reported by others.22,23 However, we observed that the transforma-
tions of two carboxylic acid groups into the corresponding NHS
esters were not complete despite using excessive amount of the
coupling reagent DIC or EDCI. The additional EDCI could lead to acti-
vation of the carboxylic acid group at the side chain of Asp residue
and related side reactions such as acylation of the free guanidine
group in the Arg residues.24 In a previous report, we demonstrated
the successful synthesis of cypate-c(RGDfK) (2) by conjugating
cypate with a protected cyclic RGD peptide, c[R(Pbf)GD(OBut)fK]
needed to improve cancer imaging, diagnosis, and therapy.
As one of the most versatile imaging modalities, optical imaging
allows noninvasive, sensitive, and real time imaging of molecular
interactions and related functions in cells, tissues, and in vivo sys-
tems. In particular, optical imaging serves as a powerful tool for
bridging the wide gap between preclinical and clinical studies,
facilitating the discovery of optimal integrin
avb3-targeted mole-
cules, and their subsequent translational studies.11–14
⇑
Corresponding author. Fax: +1 314 747 5191.
E-mail address: achilefus@mir.wustl.edu (S. Achilefu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.044

5406
Y. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5405–5409
O
O
H
NH
N
O
OH
H2N
O
N
NH
NH
HN
+
HN
N
N
O
O
HO
OH
O
O
NH2
Cypate
Cyclo[RGDfK(~)
]
+
+
N
N
N
N
H
N
NH
HO
NH
O
O
O
O
O
O
O
O
O
O
HN
O
HN
NH
NH
NH
NH
NH
NH
NH
HN
NH
HN
N
O
H2N
N
H
O
NH2
OH
H
N
H
O
O
HO
NH2
N
H
O
HO
N
H
NH
O
N
O
HN
O
HN
H
O
O
O
2
1
Fig. 1. Near-infrared fluorescent cypate and its RGD conjugates.
3. Consequently, we chose to use the same strategy for the synthe-
sis of the divalent compound 1 in this study.19 The linear protected
peptide H-D(OBut)-f-K(Dde)-R(Pbf)-G-OH was first assembled
from H-Gly-2-chlorotrityl resin using the conventional Fmoc chem-
istry, followed by cleavage of the peptide from the resin with 1%
TFA in DCM. The crude product was cyclized in the presence of Py-
BOP/HOBT/DIEA in dilute solution. The resulting cyclic protected
peptide, c[R(Pbf)GD(OBut)fK(Dde)], was treated with 2% hydrazine
in methanol to remove the Dde protecting group to form 3. Cypate
was synthesized from the benzoindo-acid and glutaconaldehyde
via its pre-acetylation intermediate in a good yield as reported pre-
viously.16,18 Cypate (1 equiv) was conjugated with 3 (3 equiv) in the
presence of EDCI (6 equiv) and HOBT (3 equiv) in DMF (Scheme 1).
The crude product, cypate-{cyclo[R(Pbf)GD(OBut)fK(ꢀ)]}2 (4), was
de-protected with TFA and purified by semi-preparative reverse
phase HPLC to afford the desired divalent RGD conjugate 1. The
compound was identified by analytical HPLC and LC–MS. As ex-
pected, 1, 2, and cypate showed similar UV–vis absorption and fluo-
based on competitive displacement against 125I-labeled echistatin.
Echistatin, an RGD-containing polypeptide isolated from viper ve-
nom, belongs to the disintegrin family of inhibitory proteins. It
binds to integrin
with linear and cyclic RGD peptides have been achieved. Previ-
ously, we determined the integrin vb3 binding affinities of some
RGD compounds using 125I-labeled echistatin tracer as reported
by Kumar et al.16,25 We also used the integrin
vb3-specific ligand
c(RGDyK) labeled with 125I at -tyrosine (y) residue instead of 125I-
avb3 with high affinity, but competitive binding
a
a
D
echistatin for the competitive integrin
a
vb3 binding assay.17 The
advantages of 125I-c[RGDyK] over 125I-echistatin include facile
labeling, good quality control, and cost effectiveness.
In the present study, the radioactivity of c(RGDyK) peptide
bound to integrin
avb3 was determined with a gamma counter.
Nonspecific binding of the tracer 125I-c(RGDyK) was determined
to be 5 to 10% of the total binding. The 50% inhibitory concentra-
tions (IC50) and affinity constant (Ki) were calculated by nonlinear
regression analysis using the GraphPad Prism 4 data fitting soft-
ware. The three RGD peptides, 1, 2, and c(RGDyK), were found to
rescence emission spectra in 20% DMSO (kmax/abs and kmax/em
:
783 nm and 811 nm, respectively).16
competitively inhibit the binding of 125I-c(RGDyK) to
avb3 in a con-
The ligand-receptor binding is a complex process that can be af-
fected by many factors, including the radioligand tracer, binding
affinity, and concentration used as well as the receptor and new li-
centration-dependent mode (Table 1). Compound 1 showed about
15 times stronger binding affinity than 2, while both c(RGDyK) and
2 exhibited comparable IC50 values. In contrast, a scrambled ana-
log, cypate-c(RGKDf), did not show significant inhibition compared
gands tested. Most of the integrin
avb3 binding assays have been
+
N
N
H
N
O
NH
O
H
N
NH
O
O
O
OBut
Pbf-HN
O
N
EDCI/HOBT/DMF
O
O
O
NH
O
NH
HN
TFA
HN
NH
HN
NH
Cypate
HN
NH
NH
1
NH
HN
O
N
O
O
Pbf-HN
N
H
O
NH-Pbf
OBut
H
O
ButO
N
O
N
H
NH
H
O
HN
O
O
NH2
O
4
3
Scheme 1. Synthesis of the divalent RGD-cypate conjugates 1.

Y. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5405–5409
5407
Table 1
The integrin
showed higher uptake than both 2 and cypate after 1 h of incuba-
tion. Nevertheless, their intracellular fluorescence reached compa-
rable levels at 6 h, suggesting that the longer incubation time
increased the non-specific uptake. These results also reflect the
a
vb3 binding of 1 and 2 traced by 125I-c(RGDyK).
Compound
IC50 (nM, Mean SD)
Ki (Mean SD)
R2
1
2
0.281 0.009
4.305 0.056
3.52 0.354
0.043 0.015
0.663 0.086
2.27 0.228
0.957
0.9874
0.9957
complex process of integrin avb3-mediated cellular internalization
c(RGDyK)
of the fluorescent divalent RGD compounds, which may be gov-
erned by multiple factors such as receptor binding, receptor
expression level, and endocytosis, as well as the structures and re-
lated physicochemical properties of the tested compounds.
We further evaluated the subcellular distribution of 1, 2, and cy-
pate by co-localization with MitoTracker Orange CM-TMRosÒ for
the mitochondria (magenta) and DRAQ5 for the nucleus (blue).
As shown in Figure 3, both 1 and 2 appear to have localized par-
tially in the nucleus and the mitochondria. These results are con-
sistent with previous reports by others.26,27
with the other RGD compounds under the same condition (data
not shown). The results further demonstrate the feasibility of using
125I-c(RGDyK) as a tracer for integrin
avb3 binding assays. Impor-
tantly, our study suggests that 125I-c(RGDyK) is a reliable tracer
for competitive binding assays of new compounds containing
RGD structural motif with similar binding features and comparable
binding affinity. Furthermore, 125I-c(RGDyK) may serve as a sensi-
To investigate integrin
a
vb3 targeting and tumor localization in
M) were intravenously injected by
tive method for studying some integrin avb3 ligands that could not
vivo, both 1 and 2 (100 L, 60
l
l
compete favorably against 125I-echistatin. We will further study
the generality of 125I-c(RGDyK) and compare it with 125I-echistatin
tail vein into nude mice bearing A549 tumor xenografts (5–7 mm
diameter). Figure 4A shows the results of NIR fluorescence imaging
(excitation and emission at 780 nm and 830 nm, respectively) at
1 h, 4 h, and 24 h post-injection. Compound 1 showed tumor local-
ization in nude mice with good contrast between the tumor and
non-target tissues at 4 h post-injection. In contrast, 2 had high
accumulation in the liver and kidneys with low tumor uptake.
Fluorescence intensity measurement shows that 1 had higher tu-
mor/muscle ratio at 4 h and 8 h than 2 (Fig. 4B). After euthanizing
the animals at 24 h post-injection, the ex vivo organ analysis
showed the relatively high uptake of 1 in the tumor and the liver
compared with the kidneys, heart, and other tissues (Fig. 4C and
4D). The results are in good agreement with the in vivo noninva-
sive optical imaging results.
for studying integrin
NIR fluorescence microscopy was used to evaluate the com-
pounds for integrin vb3-mediated cellular binding and uptake.
This also allowed the imaging of 1, 2, and cypate in integrin vb3
positive A549 cells at different time points. The cell staining results
are dependent on the incubation time (Fig. 2). Compound 1
avb3-binding in future work.
a
a
Divalent and multivalent approaches have been widely studied
in molecular discovery for improving the selectivity, specificity,
and potency of receptor binding as well as the related biological
and pharmacological activities.16,28–32 Clearly, increased local con-
centration and divalent or multivalent interaction can enhance the
integrin
avb3 binding affinity of RGD compounds. Various divalent
and multivalent RGD peptides for integrin
a
vb3-targeted imaging
such as 18F-FPPRGD2 and some fluorescent labeled RGD com-
pounds have also been reported for tumor targeted imaging, ther-
apy, and drug delivery.14,16,26,32–37 Especially, it is important to
have the suitable linkage between two RGD motifs that can span
the two neighboring integrin
avb3 sites for simultaneous binding.
Fig. 2. Binding and internalization of 1, 2, and cypate in A549 cells at different time
points.
As shown above, 1 represents a new type of NIR fluorescent diva-
Fig. 3. Subcellular distribution of 1 and 2 at 16 h of incubation. Fluorescent probes were excited (at 488 nm, 633 nm and 755 nm) and two-dimensional confocal images
acquired by scanning a field at 2 s per pixel. Each image shows overlay of nuclear stain (blue), mitochondrial stain (green) and cypate (red). Arrows point to cypate
fluorescence indicative of the presence of the compound in the marked region.
l

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Y. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5405–5409
lent ligands using cypate as both linker and probe. Unlike other
flexible linkers, the cypate motif consists of ploymethines and ben-
zoindoline rings, which might help to pre-organize the divalent
grins with decreased binding entropy and enhanced stability of
the resulting ligand-receptor complex.
By comparing with 2 and cypate, the significant improvement
RGD ligand for simultaneous binding of two adjacent
avb3 inte-
of 1 in tumor localization suggests the enhanced integrin avb3-
Fig. 4. Optical imaging of 1 and 2 in mice. (A) In vivo optical imaging of tumors (circled areas) at 1, 4, and 24 h post –injection; (B) time-dependent tumor uptake; (C) ex vivo
biodistribution of fluorescence at 24 h post-injection; and (D) ex vivo fluorescence imaging of representative organs.
27 atoms
+
N
N
H
N
NH
O
O
O
O
O
O
NH
O
HN
NH
HN
NH
NH2
HN
NH
NH
N
H
O
N
O
H2N
HO
OH
H
O
N
H
O
N
HN
NH
O
H
O
O
Cell surface
Fig. 5. The two RGD motifs linked by cypate for simultaneous binding of two avb3 integrins.

Y. Ye et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5405–5409
5409
targeting for tumor optical imaging. The results also support our
hypothesis that the divalent compound can simultaneously bind
to two adjacent integrin vb3 proteins and exhibit synergistic ef-
fects on receptor- and tumor-targeting. Furthermore, the linker
of a divalent ligand plays an important role in achieving significant
synergistic effects.4,31 The 27-atom length between the two RGD
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