αvβ3-Integrin-targeting lanthanide complex: Synthesis and evaluation as a tumor-homing luminescent probe

Article abstract of DOI:10.1016/j.bmcl.2011.05.001

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α_vβ ₃-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α_vβ ₃-integrin-expressing U87-MG cells were successfully obtained by employing the probes.

Full text of DOI:10.1016/j.bmcl.2011.05.001

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3515–3518
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
avb3-Integrin-targeting lanthanide complex: Synthesis and evaluation
as a tumor-homing luminescent probe
⇑
⇑
Takeo Ito , Masaki Inoue, Kanako Akamatsu, Eriko Kusaka, Kazuhito Tanabe, Sei-ichi Nishimoto
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has
emerged in the last few decades, providing high-contrast images of cells through detection of the delayed
emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic
peptide c(RGDfK) to visualize the avb3-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the
macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating
Received 5 April 2011
Revised 29 April 2011
Accepted 2 May 2011
Available online 5 May 2011
that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of
Keywords:
avb3-integrin-expressing U87-MG cells were successfully obtained by employing the probes.
Cyclic RGDfK peptides
Time-resolved fluorescence imaging
U87-MG
Ó 2011 Elsevier Ltd. All rights reserved.
Macrocyclic compound
Lanthanide complexes
Luminescence from trivalent lanthanide ions is characterized by
narrow-band emission spectra, long lifetimes (of the order of milli-
seconds), and a large gap between the excitation and emission
bands. Because of their long emission lifetimes, it is possible to carry
out time-resolved detection of the complexes in order to eliminate
background signals and autofluorescence from biomolecules.1–6
Time-resolved fluoroimmunoassay has been well established as
one of the applications of such lanthanide complexes.7,8 For the
improvement of the luminescence intensity of lanthanide com-
plexes, it is desirable for the ligand structure to show intense absorp-
tion in the UV–vis wavelength region, and to exhibit efficient energy
transfer from the excited state of the ligand to the coordinated lan-
thanide ion. In addition, the ligand should form a strong complex
with the lanthanide ion and shield the metal ion from nonradiative
deactivation by solvent molecules.
In the present study, we synthesized trivalent lanthanide com-
plexes of macrocyclic bipyridine ligands bearing cyclic peptides for
the visualization of tumor cells expressing integrins on their sur-
faces. For the purposes of optical imaging of tumor cells and their
functions, appropriate functionalization of the ligand structure is
necessary, but such modifications often alter the luminescence
properties. Integrins are transmembrane glycoproteins consisting
esis, and thus the expression of this integrin is correlated with
tumor aggressiveness. Cyclic peptides containing the RGD (Arg-
Gly-Asp) sequence are well known as specific ligands of the vb3
integrin cell-surface receptor. The ability to visualize and quantify
the vb3 integrin expression level will provide new opportunities
for following integrin expression in tumor tissues.13–17 As reported
below, we introduced cyclic (Arg-Gly-Asp- -Phe-Lys) [c(RGDfK)]
into a lanthanide complex for molecular imaging of integrin-
expressing tumor cells, and investigated the effects of the function-
alization of the ligand structure on the luminescence properties.
a
a
D
The aims of the present study are to develop a novel avb3-inte-
grin-targeting fluorescent probe, and to investigate the effects of
side chains for the introduction of c(RGDfK)18,19 peptides on its
fluorescence properties. It has been demonstrated that bipyri-
dine-cryptand ligands can enhance the fluorescence lifetimes of
their lanthanide complexes by shielding the trivalent lanthanide
of
a
and b subunits that mediate cell–cell and cell-to-extracellular
vb3 appears to
matrix interactions.9–12 In particular, the integrin
a
play an important role in tumor growth, metastasis, and angiogen-
⇑
Corresponding authors. Tel.: +81 74 383 7054; fax: +81 75 383 2504 (T.I.).
E-mail addresses: takeoit@scl.kyoto-u.ac.jp (T. Ito), nishimot@scl.kyoto-u.ac.jp
Figure 1. Structure of c(RGDfK)-macrocyclic bipyridine ligand (1).
(S. Nishimoto).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.001

3516
T. Ito et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3515–3518
ions from water molecules.20–23 We designed a macrocyclic bipyr-
idine ligand bearing two c(RGDfK) peptides as tumor-targeting
moieties (1, Fig. 1). The macrocyclic compound 2 was synthesized
from 6,60-dimethyl-2,20-bipyridine in three steps, following a pre-
viously reported procedure.24,25 For the conjugation of the
c(RGDfK) peptide, ligand 2 was derivatized to form the dicarbox-
ylic acid 3, and then reacted with N-(2-aminoethyl)maleimide to
afford ligand 4 (Scheme 1). Coupling between the thiol-derivatized
c(RGDfK) peptide [c(RGDfK)-SH] and 4 yielded 1 and the monosub-
stituted ligand 5. Trivalent europium (Eu3+) and terbium (Tb3+
)
complexes of 1, 3, and 5 were prepared by adding lanthanide chlo-
rides (LnCl3) to solutions of the corresponding ligands.
Figure 2 shows the UV absorption spectra of Eu3+ꢀ1, 3 in H2O.
The absorption band at around 310 nm is mainly due to the bipyr-
idine structure of the ligands. Fluorescence spectra of the com-
plexes Ln3+ꢀ1 (Ln3+ = Eu3+ and Tb3+
) were obtained upon
excitation at 315 nm, and showed characteristic luminescence
spectra arising from the emissive 5D0 level to the ground-state
7FJ (J = 0–4) level of Eu3+, or from the 5D4 level to the 7Fj (J = 3–6)
level of Tb3+ (Fig. 3). It has been well established that light-harvest-
ing ligands absorb UV light and transfer energy from the excited
states to the coordinated lanthanide ions.26,27 In fact, other com-
plexes (Ln3+ꢀ3, 5) showed quite similar fluorescence spectra (data
not shown). In order to gain further understanding of the energy-
transfer process in our complexes, we estimated the energy level
of the excited triplet state of 3 from its phosphorescence spectrum
obtained at 77 K (Supplementary data, Fig. S1). Phosphorescence
Figure 2. UV absorption spectra of Eu3+ꢀ1 (solid line) and Eu3+ꢀ3 (dashed line);
concentration 10 lM in H2O.
from ligand
3 showed a maximum at around 480–490 nm
(20800–20400 cmꢁ1), and was efficiently quenched in the case of
the corresponding Eu3+ complex (Eu3+ꢀ3), which suggests that
the emission process involves energy transfer from the excited
triplet state of the ligand to Ln3+
.
The quantum yields (UF) and lifetimes (s) of the emission are
listed in Table 1. Emission quantum yields obtained from the com-
plexes are relatively low, because back electron transfer deactiva-
tion from the excited Ln3+ to the triplet energy levels of the
ligands is likely to occur due to small band gaps between them.
The hydration number (the number of water molecules, q) of the
inner coordination sphere of the lanthanide ions can be estimated
by measuring the differences between the emission lifetimes in
Figure 3. Steady-state emission spectra of Eu3+ꢀ1 (10
l
M, solid line) and Tb3+ꢀ1
(0.5 lM, dashed line) in H2O. Complexes were photoexcited at 315 nm (excitation
and emission slit widths: 5 nm, respectively).
Scheme 1. Reagents and conditions: (a) NCS, BPO, CCl4, reflux, 24 h, 25%; (b) TsNHNa, DMF, 80 °C, 5 h; (c) conc. H2SO4, 110 °C, 2 h, 30% in two steps; (d) ethyl bromoacetate,
K2CO3, CH3CN/CH2Cl2, reflux, 24 h; 61%; (e) KOH, EtOH, rt, 3 h, 70%; (f) N-(2-aminoethyl)maleimide, HCTU, Et3N, CH3CN, rt, 6 h, 30%; (g) c(RGDfK)-SHCOCH3, NH2OHꢂHCl,
EDTA-HEPES buffer, rt, 4 h.

T. Ito et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3515–3518
3517
Table 1
Fluorescence lifetimes and quantum yields of the complexes
Ligand
Ln3+
1
3
5
Eu3+
Tb3+
Eu3+
Tb3+
Eu3+
Tb3+
sH2O (ms)
sD2O (ms)
sPBS (ms)
qa,b
0.8
1.6
0.7
0.4
0.05
1.9
2.5
1.8
0.5
0.17
0.5
1.0
0.6
0.8
0.01
0.8
1.0
0.9
0.8
0.17
0.7
1.1
0.9
0.2
0.03
1.0
1.3
1.2
0.8
0.06
c
UF
Fluorescence lifetimes were measured for complexes 1, 3, and 5 in H2O, D2O, and
phosphate buffer saline (PBS).
a
q(Eu) = 1.11 ꢄ (1/sH2Oꢁ1/sD2Oꢁ0.31), Ref. 29.
b
q(Tb) = 5.0 ꢄ (1/sH2Oꢁ1/sD2Oꢁ0.06), Ref. 30.
c
Measured in H2O.
H2O and D2O using the equations of Horrocks and Parker.28–30 The
fluorescence lifetimes of the complexes were determined to be
ꢃ1 ms, which should be long enough for the time-resolved fluores-
cence imaging of biomolecules to discriminate the fluorescence of
these complexes from autofluorescence originating from the tis-
sue. The very slight shortening of their lifetimes observed in phos-
phate buffer encouraged us to use the probes for the microscopic
observation of biomolecules under physiological conditions. The
q-values obtained for the Eu3+ and Tb3+ complexes are 0.2–0.8,
which suggests that there is less than one molecule of water in
the first coordination sphere. The fluorescence lifetime and q-value
for Eu3+ꢀ2 were also obtained as 0.2 ms and 3.2 (in H2O), respec-
tively. Thus, the carbonyl groups on the linkers of Ln3+ꢀ3 should
contribute to the coordination of Eu3+. Considering that Ln3+ꢀ1
and Ln3+ꢀ3 showed similar q-values, the c(RGDfK) residues may
not be directly coordinated to the central lanthanide ion. It is note-
worthy that the lifetime of Tb3+ꢀ1 in H2O (1.9 ms) is rather longer
than those of other Tb3+ complexes (0.8–1.0 ms), probably because
the c(RGDfK) peptides of Tb3+ꢀ1 prevent deactivation by the clo-
Figure 4. Microscopic images of live PC-3 (a, b) and U87-MG (c–f) cells incubated
with (a–d) Eu3+ꢀ1 (100
l
M), and (e, f) Eu3+ꢀ3 (100
lM). (a, c, e) Phase-contrast
sely diffusing water molecules around Tb3+ 29,30 Unlike the Eu3+
.
images, and (b, d, f) merged images of fluorescence and phase-contrast pictures.
complexes, emission from the Tb3+ complexes (UF = 0.17) is in-
tense enough for cell imaging. This is partly because of smaller en-
ergy gap between 5D0 and 7F6 states of Eu3+ than that between 5D4
and 7F0 of Tb3+ enhances non-radiative deactivation of the excited
c(RGDfK) units are not sufficiently well separated to bind two
neighboring
In summary, we have synthesized c(RGDfK)-linked macrocyclic
bipyridine as ligands for trivalent lanthanide ions (Eu3+ and Tb3+
in order to utilize them as time-resolved fluorescence probes for
vb3-integrin-expressing tumor cells. It has been demonstrated
a
vb3 integrin sites simultaneously.13
states of Eu3+ 8,19
.
The cytotoxicity of complexes Eu3+ꢀ1 and Tb3+ꢀ1 was assayed
by the WST-8 method (Fig. S2).31 Both probes induced cell aggrega-
tion, but showed no remarkable toxicity in the concentration range
)
a
M). The stability of Eu3+ꢀ1 and Tb3+ꢀ1
that the introduction of c(RGDfK) units into the bipyridine macro-
cycle has little effect on the fluorescence properties, because the
bipyridines and the linkers shield the central lanthanide ion from
c(RGDfK) and solvent molecules. Successful visualization of the
target tumor cells has encouraged us to develop novel time-re-
solved fluorescence probes that can be photoexcited by lower-en-
ergy UV light than that employed in this study.
below 100
l
M (IC50 >100
l
was evaluated during a period of 42 h at 37 °C in the medium. By
emission analysis it was found that both complexes were suffi-
ciently stable enough for the microscopic observation (Fig. S3).
Fluorescence imaging of living tumor cells was examined by incu-
bating the probes with highly
avb3-integrin-expressing human
glioblastoma U87-MG cells or with less expressing human prostate
cancer PC-3 cells at 37 °C. As shown in Figure 4, the U87-MG cells
were successfully visualized by Eu3+ꢀ1 upon excitation at
330 40 nm and fluorescence collection at 605 28 nm (Fig. 4c,
d). On the other hand, only very weak fluorescence was observed
from PC-3 cells incubated with Eu3+ꢀ1 (Fig. 4a, b) and U87-MG
cells incubated with Eu3+ꢀ3 (Fig. 4e, f). These results clearly sug-
gest that the lanthanide complex Eu3+ꢀ1 recognizes the integrin
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.001.
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