Dual-targeting conjugates designed to improve the efficacy of radiolabeled peptides

Article abstract of DOI:10.1039/c2ob26127h

Radiolabeled regulatory peptides are useful tools in nuclear medicine for the diagnosis (imaging) and therapy of cancer. The specificity of the peptides towards GPC receptors, which are overexpressed by cancer cells, and their favorable pharmacokinetic profile make them ideal vectors to transport conjugated radionuclides to tumors and metastases. However, after internalization of the radiopeptide into cancer cells and tumors, a rapid washout of a substantial fraction of the delivered radioactivity is often observed. This phenomenon may represent a limitation of radiopeptides for clinical applications. Here, we report the synthesis, radiolabeling, stability, and in vitro evaluation of a novel, dual-targeting peptide radioconjugate designed to enhance the cellular retention of radioactivity. The described trifunctional conjugate is comprised of a Tc-99m SPECT reporter probe, a cell membrane receptor-specific peptide, and a second targeting entity directed towards mitochondria. While the specificity of the first generation of dual-targeting conjugates towards its extracellular target was demonstrated, intracellular targeting could not be confirmed probably due to non-specific binding or hindered passage through the membrane of the organelle. The work presented describes a novel approach with potential to improve the efficacy of radiopharmaceuticals by enhancing the intracellular retention of radioactivity.

Full text of DOI:10.1039/c2ob26127h

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PAPER
Dual-targeting conjugates designed to improve the efficacy of radiolabeled
peptides†
Christiane A. Kluba, Andreas Bauman, Ibai E. Valverde, Sandra Vomstein and Thomas L. Mindt*
Received 12th June 2012, Accepted 1st August 2012
DOI: 10.1039/c2ob26127h
Radiolabeled regulatory peptides are useful tools in nuclear medicine for the diagnosis (imaging) and
therapy of cancer. The specificity of the peptides towards GPC receptors, which are overexpressed by
cancer cells, and their favorable pharmacokinetic profile make them ideal vectors to transport conjugated
radionuclides to tumors and metastases. However, after internalization of the radiopeptide into cancer
cells and tumors, a rapid washout of a substantial fraction of the delivered radioactivity is often observed.
This phenomenon may represent a limitation of radiopeptides for clinical applications. Here, we report the
synthesis, radiolabeling, stability, and in vitro evaluation of a novel, dual-targeting peptide radioconjugate
designed to enhance the cellular retention of radioactivity. The described trifunctional conjugate is
comprised of a Tc-99m SPECT reporter probe, a cell membrane receptor-specific peptide, and a second
targeting entity directed towards mitochondria. While the specificity of the first generation of dual-
targeting conjugates towards its extracellular target was demonstrated, intracellular targeting could not be
confirmed probably due to non-specific binding or hindered passage through the membrane of the
organelle. The work presented describes a novel approach with potential to improve the efficacy of
radiopharmaceuticals by enhancing the intracellular retention of radioactivity.
cells occurs at a rate comparable to the internalization.5–8 Also, a
Introduction
significant washout of radioactivity from tumors over time is
often observed in vivo.6,9–11 This phenomenon can render the
Conjugation of radionuclides to tumor-targeting peptides enables
their application for the diagnosis (using gamma- or positron-
emitting nuclides) and therapy (employing beta- or alpha-particle
emitters) of cancer. In particular, radiometal-labeled regulatory
peptides, which target G-protein-coupled receptors (GPCRs)
overexpressed in different cancers, have been a focus of radio-
pharmaceutical and nuclear medicinal research over the past
decade. As a result, some radiometal-labeled peptides have
found routine application in the clinic (e.g., somatostatin deriva-
tives for the management of neuroendocrine tumors), while
other promising candidates are currently the subject of preclini-
cal evaluation or have recently advanced to clinical trials.1–4
Radiolabeled regulatory peptides internalize into tumor cells
by GPCR-mediated endocytosis. A potential drawback of a
number of reported radiopeptides is represented by a rapid
washout of a substantial fraction of the intracellular radioactivity
from cancer cells (in vitro) and tumors (in vivo). In some
instances, the in vitro externalization of radioactivity from cancer
initial efforts of delivering specifically radioactive nuclides to
cancer cells and tumors in part futile. The mechanism by which
the radioactivity is externalized from cancer cells and tumors is
yet not fully understood and can vary depending on, e.g., the
peptide carrier or the composition of the radioconjugate
employed. For example, cellular washout of radioactivity in the
form of metabolic degradation products has been reported for
radiometal-labeled derivatives of bombesin and minigastrins.12,13
On the other hand, the externalization of intact radiometal conju-
gates has been observed for somatostatin and exendin-4 deriva-
tives.7,9,11 Regardless of the metabolic fate of the tumor-
targeting peptide vector, it is generally accepted that the efficacy
of radioconjugates correlates with a high rate of internalization
into, and a low rate of externalization from targeted tissue.
We herein wish to report our efforts to combine tumor-
targeting radiolabeled peptides with a second moiety which
targets intracellular components. We hypothesized that such an
additional moiety (e.g., directed against intracellular proteins,
enzymes, or cell compartments) of the resulting dual-targeting
radioconjugates will decrease the externalization rate of radio-
activity from cancer cells and tumors independent of the vector
used. An increased retention time of the delivered radioactivity
within tumors holds the promise to improve the signal-to-back-
ground ratio important for diagnostic (imaging) applications and
University of Basel Hospital, Department of Radiology and Nuclear
Medicine, Division of Radiological Chemistry, Petersgraben 4,
4031 Basel, Switzerland. E-mail: TMindt@uhbs.ch;
Fax: +41 (0)61 265 55 99; Tel: +41 (0)61 556 53 80
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26127h
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to enhance the efficacy of therapeutic radionuclides as a result of
the increased energy deposited in cancerous tissue.
by various cancer cell types including prostate, breast, and
small-cell lung cancer.30 The potential of derivatives of [Nle14]-
BBS(7–14) radiolabeled with the 99mTc(CO)3 core for cancer
imaging has been demonstrated in vitro and in vivo.12,15,28 Based
on literature precedence, we introduced a spacer made of mul-
tiple β-alanine units to separate the tumor-targeting peptide from
other parts of the conjugate in order to minimize potentially
unfavorable interferences.10,12 As an intracellular target, we
chose the organelle mitochondria. It has been shown that lipo-
philic, cationic species such as triphenylphosphonium (TPP)
moieties can be driven electrophoretically through negatively
charged transmembranes, resulting in an accumulation of the
compounds inside the energized mitochondria of cells. TPPs
have been used as a drug delivery system for the transport of
therapeutic agents to the mitochondria.31 Also, radioactive
labeled TPP derivatives have been studied for myocardial
imaging and tumor imaging.32
In recent years, new approaches for the development of radio-
pharmaceuticals have emerged, which aim at the combination of
the tumor-targeting properties of radiolabeled peptides with
other entities of interest. Reported examples include the combi-
nation of radiopeptides with pharmacological modifiers to
improve their pharmacokinetic and/or –dynamic charac-
teristics14–16 or therapeutic efficacy,7,17–19 optical dyes for the
design of dual-modal imaging probes for high sensitivity and
high resolution imaging,20,21 and therapeutic agents22 for the
development of theranostics.23 The potential of multifunctional
radioconjugates, which exploit the individual properties of com-
ponents or synergistic effects thereof, respectively, for appli-
cations in nuclear medicine is intriguing, however, their
synthesis can be challenging.
We have previously reported a modular synthetic approach
which employs the Cu(I)-catalyzed alkyne–azide cycloaddition
(CuAAC; click chemistry)24,25 for the efficient preparation of
multifunctional radioconjugates.14,26 In this approach, an N(α)-
propargyl derivative of the amino acid lysine functions as the
center piece of the final conjugate by providing the means for
conjugation of two different chemical/biological moieties by
selective amide bond formation and CuAAC (Fig. 1). In
addition, the N(α)-propargyl lysine derivative is a prochelator
which forms, after formation of the 1,2,3-triazole by CuAAC, a
tridentate ligand system for the stable complexation of the
99mTc-tricarbonyl core suitable for imaging by single photon
emission computed tomography (SPECT).27,28 The diagnostic
isotope technetium-99m forms a “matched pair” with chemically
related therapeutic β−-particle emitters rhenium-186/188 and,
therefore, the chemistry developed has the potential to be used
for the development of radioconjugates for both diagnostic and
therapeutic applications in nuclear medicine.29
The combination of receptor-targeting radiopeptides with moi-
eties specific for the cell nucleus (e.g., DNA intercalator or cell
penetrating peptides) has been reported to exploit potentially the
therapeutic effect of short-range Auger electron-emitting radio-
nuclides, however with varying degrees of success.7,19 The use
of mitochondria-specific entities (e.g., TPPs) for improving the
intracellular retention of radioactivity after selective delivery by
tumor-avid peptides has yet not been described in the context of
dual-targeting radioconjugates.
Results and discussion
Scheme 1 outlines the synthesis of building blocks required for
the assembly of trifunctional conjugates. The preparation of the
We set out to investigate the use of the “click chemistry” strat-
egy for the assembly of trifunctional conjugates made up of a
99mTc-tricarbonyl reporter probe for SPECT, a tumor-targeting
peptide, and an additional entity specific for an intracellular
target, e.g., a cellular compartment. For the first generation of the
proposed dual-targeting radioconjugates, we selected a reported,
stabilized variation of the minimal binding sequence of bombe-
sin (BBS), [Nle14]BBS(7–14),12 as an extracellular tumor-target-
ing component. BBS is a regulatory peptide that targets the
gastrin-releasing peptide (GRP) receptor, which is overexpressed
Fig. 1 Assembly of trifunctional conjugates by selective amide bond
formation, CuAAC (click chemistry), and (radio)metal complexation;
residues R1 and R2 represent two different moieties of biological func-
tion to be combined in the final (radio)metal conjugate.
Scheme 1 Synthesis of building blocks. (a) SPPS: (i) piperidine (20%)
in DMF; TBTU–HOBt (4 equiv.), (ii) Fmoc-amino acids or azidoacetic
acid (3 equiv.), HATU (3 equiv.); (b) see ref. 14; (c) toluene, reflux, 5 h.
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tumor targeting peptide [Nle14]BBS(7–14), its N-terminal
elongation with three β-Ala units, and azidoacetic acid was
accomplished by standard solid phase peptide synthesis (SPPS)
yielding protected azide-functionalized peptide 1 on the resin
(Scheme 1A; see the Experimental section for details). The
central piece of the trifunctional conjugate, N(α)Boc-N(α)propar-
gyl lysine 3, was prepared from commercial BocLysOMe (2) in
five steps as previously described (Scheme 1B).14 An appropri-
ately functionalized mitochondria-targeting entity, 3-tris(p-
methoxyphenyl)-phosphonium propionic acid 6, was synthesized
by the reaction of tris(p-methoxyphenyl)phosphine (4) with
3-bromo-propionic acid (5; Scheme 1C).33
The assembly of the trifunctional conjugate started with the
coupling of TPP derivative 6 to the N(ε)-amine of the side chain
of Lys-derivative 3 followed by TFA-mediated removal of the
N(α)Boc protective group (Scheme 2). The resulting intermedi-
ate 7 was then reacted with protected, azide-functionalized
peptide 1 by CuAAC on a solid support. After cleavage from the
resin and deprotection, conjugate 9 was purified by preparative
HPLC and its structure was confirmed by mass spectrometric
analysis (Table 2). To study the effect of the mitochondria-target-
ing TPP moiety, we also synthesized reference compound 10
from intermediate 814 by the same synthetic pathway (see the
Experimental part for details). Compound 10 is identical to con-
jugate 9 in all respects but lacks the TPP moiety at the N(ε)-
amine of the Lys residue (replaced by an acetate group).
the desired radiopeptides [99mTc(CO)3(L)] (L = 9, 10) in >95%
radiochemical yield and purity. The radioconjugates were
obtained with a specific activity of up to 17.4 GBq μmol−1 (not
optimized). In the case of methyl ester 10, ester hydrolysis and
labeling with 99mTc-tricarbonyl occurred simultaneously under
the reaction conditions applied.14 For characterization purposes,
the corresponding non-radioactive rhenium tricarbonyl com-
plexes [Re(CO)3(L)] (L = 9, 10) were prepared by the reaction of
34
conjugates 9 and 10 with [Re(CO)3Br3][Et4N]2 for 60 min at
100 °C and analyzed by mass spectrometry. The identity of
[
99mTc(CO)3(L)] (L = 9, 10) was confirmed in each case by com-
parison of the γ-HPLC trace with the UV trace of the corres-
ponding rhenium complexes [Re(CO)3(L)] (L = 9, 10), a
procedure which is common practice with 99mTc complexes on
an n.c.a. (no carrier added) level (Fig. 2).
The stability of the radiolabeled peptides was evaluated in
different media at 37 °C. Both [99mTc(CO)3(L)] (L = 9, 10) were
stable (>95% as determined by HPLC) in PBS (pH 7.4), NaCl
(0.9%), and in a cell culture medium (1% FBS) for >4 h, the
period of time required for the in vitro experiments described
below (see the Experimental part for details). We also verified
the stability of the Tc-99m complexes in a ligand challenge
experiment.35 Thus, [99mTc(CO)3(L)] (L = 9, 10) were incubated
at 37 °C in PBS containing a 4500-fold excess of histidine, an
excellent tridentate chelator for complexation of the Tc-99m
tricarbonyl core. No transmetallation was observed over a period
of 24 h, therefore demonstrating the suitability of the employed
Lys-based chelator unit of the conjugates.
Both peptide conjugates 9 and 10 were readily radiolabeled
with [99mTc(CO)3(H2O)3]+ within 30 min at 100 °C, providing
Scheme 2 Assembly of dual targeting radiopeptide conjugates. (a) For
7: 6, SOCl2, CH2Cl2, rt overnight; then 3 and Et3N, −78 °C, 4 h; for 8:
see ref. 14; (b) CH2Cl2–TFA (3 : 1), rt overnight; (c) DIPEA, [Cu-
(CH3CN)4]PF6, TBTA, DMF, rt overnight; (d) trifluoroacetic acid,
phenol, water, triisopropylsilane (87.5/5/5/2.5%), 2 h, rt; (e) for M = Tc-
99m: 0.1 mM of 9 or 10 in water, [99mTc(CO)3(H2O)3]+ (100–
250 MBq), 100 °C, 30 min; for Re complexes: 0.4 mM of 9 or 10 in
water, [Re(CO)3Br3][Et4N]2 (1.5 equiv.), 100 °C, 60 min.
Fig. 2 HPLC chromatographs of peptide precursor 9 (UV-trace A), the
corresponding metal conjugates [Re(CO)3(9)] (UV-trace B), and [99mTc-
(CO)3(9)] (γ-trace C). The small difference of retention times between
[Re(CO)3(9)] and [99mTc(CO)3(9)] (traces B and C) is due to the serial
arrangement of the UV- and γ-detectors.
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We next investigated in vitro the receptor affinity as well as
internalization and externalization characteristics of the conju-
gates [99mTc(CO)3(L)] (L = 9, 10) using PC-3 cells (n = 3–4 in
triplicate). The binding affinity of the bombesin derivatives
towards GRP-receptors was determined by receptor saturation
binding experiments employing increasing amounts of the radio-
conjugates (Fig. 3). Non-specific receptor binding was assessed
by using an excess of natural bombesin BBS(1–14). Reference
peptide [99mTc(CO)3(10)] displayed a dissociation constant (Kd)
of 5.6 0.8 nM. In comparison, dual-targeting [99mTc(CO)3(9)]
exhibited a decreased receptor binding affinity with a Kd of
41.6 5.8 nM. Values of dissociation constants in the single- to
lower double-digit nanomolar range are usually reported for
radiolabeled regulatory peptides.
Despite the differences in receptor binding affinities, we
observed that both radioconjugates internalized into PC-3 cells
to the same extent and at a comparable rate (approx. 25% of the
applied radioactivity within 30–90 min; Fig. 4). These results are
comparable with those reported for related BBS derivatives
labeled with the 99mTc-tricarbonyl core.15 In comparison to refer-
ence compound [99mTc(CO)3(10)], [99mTc(CO)3(9)] exhibited
some degree of non-specific internalization as determined by
blocking experiments using a 1000-fold excess of natural
bombesin BBS(1–14). Albeit not ideal, low levels of non-
specific binding and internalization have been described for a
number of receptor-specific radiotracers, some of which have
advanced to the stage of preclinical or clinical investigations
nevertheless.11,36 In the case reported herein, however, the
lipophilicity introduced by the TPP moiety and/or the 99mTc-
(CO)3 core could also be in part responsible for the observed
non-specific binding and internalization of the trifunctional
conjugate.
Determination of the log D of peptide precursors 9 and 10,
and the corresponding metal conjugates by HPLC indeed
revealed increased lipophilicities for TPP-containing peptide 9
and, more pronounced, its metal-labeled analogues [M(CO)3(L)]
(M = 99mTc, Re; L = 9) (Table 1, n = 3). While some non-
specific binding and internalization of a peptidic radiotracer is
not necessarily a criterion for exclusion from further develop-
ments, one has to critically acknowledge that an increased lipo-
philicity can result in an unfavorable biodistribution. On the
other hand, it is known that the lipophilicity of a radiopeptide
can be adjusted by the attachment of polar groups (e.g., by pegy-
lation or glycosylation), or the use of charged and/or polar
linkers.16,37
Experiments performed with radiopeptides [99mTc(CO)3(L)]
(L = 9, 10) showed, undesirably, a very similar externalization
pattern for both conjugates, resulting in a washout of ca. 50% of
intracellular radioactivity after 90 min, approximately the same
time required for specific internalization of the radioconjugates
into GRP-receptor expressing PC-3 cells (Fig. 5).
Fig. 4 Internalization of radiometal conjugates in GRP-receptor
expressing PC-3 cells; normalized to 106 cells per well; total binding of
[
99mTc(CO)3(10)] (○, dotted line) and [99mTc(CO)3(9)] (Δ, continuous
line); non-specific binding as determined by blocking experiments in the
presence of excess natural BBS: [99mTc(CO)3(10)] (●, dotted line) and
[
99mTc(CO)3(9)] (▲, continuous line).
Table 1 Log
D
of peptides and (radio)metal-labeled analogues
determined by HPLC
Compound
Log D
9
1.23 ( 0.04)
3.01 ( 0.01)
3.11 ( 0.02)
0.51 ( 0.06)
0.80 ( 0.07)
0.78 ( 0.01)
[Re(CO)3(9)]
Fig. 3 Receptor saturation binding experiments of radiometal conju-
gates using GRP-receptor expressing PC-3 cells; normalized to 106 cells
per well; specific binding of [99mTc(CO)3(10)] (●, dotted line) and
[
99mTc(CO)3(9)]
10
[Re(CO)3(10)]
99mTc(CO)3(10)]
[
99mTc(CO)3(9)] (▲, continuous line); Kd values were determined by
[
nonlinear regression.
Table 2 Yields and analytical data of synthesized peptides
Compound
Molecular formula
Yield (isolated)
Calcd MW (g mol−1
)
MALDI-MS m/z
Purity (HPLC)
9
10
C88H123N21O19
C67H103N21O16
P
25.9%
23.4%
1808.90
1457.79
[M]+: 1808.86
98.6%
97.6%
[M + H]+: 1459.2, [M + 2H]2+: 730.2
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of quantification of the binding of a radiotracer to isolated
mitochondria.
In a further effort to verify the specificity of [99mTc(CO)3(9)]
towards mitochondria, we performed experiments following pro-
cedures described by Murphy et al. and others,38–40 in which
isolated mitochondria are pretreated with established reagents
that either disrupt the transmembrane potential of the organelle
(sodium azide (NaN3) or carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP)) or enhance it (succinate), respectively.
In all cases, the binding of [99mTc(CO)3(9)] to mitochondria was
not influenced by the presence of the additive (Fig. 6). It is there-
fore possible that the binding of [99mTc(CO)3(9)] to mitochon-
dria described above is not driven by the transmembrane
potential of the organelle but may be the result of non-specific
binding. Alternatively, the ability of TPPs to shuttle conjugated
cargo into mitochondria can vary depending on the composition
of the conjugate. For instance, examples for successful and
unsuccessful mitochondria-targeting of TPP moieties conjugated
to (not receptor specific) peptides have both been reported.38,41
Thus, it is also feasible that the passage of [99mTc(CO)3(9)]
through the membrane of mitochondria is hindered and therefore
interfered with the intracellular targeting. Distinction of the two
possible processes, which could impede the specific accumu-
lation of the dual-targeting radioconjugate in mitochondria, may
be challenging and requires further investigations. In the mean-
time, we are examining a second generation of dual-targeting
radioconjugates, which target cytosolic proteins instead of orga-
nelles and therefore do not require trafficking through intracellu-
lar membranes. By simplifying the intracellular targeting system
we expect to gain more insights into the structural requirements
of a dual-targeting radioconjugate leading to an improved reten-
tion of radioactivity in cancer cells and tumors.
Fig. 5 Externalization of radioactivity from GRP-receptor expressing
PC-3 cells. Externalization of the total internalized fraction at t = 0
(100%) of radiotracers: [99mTc(CO)3(10)] (●, dotted line) and [99mTc-
(CO)3(9)] (▲, continuous line).
Experimental
CAUTION: 99mTc is a γ-emitter (140 keV) with a half-life of
6.01 h. All reactions involving 99mTc were performed in a labo-
ratory approved for the handling of radionuclides and appropriate
safety procedures were followed at all times to prevent
contamination.
Fig. 6 Accumulation of radiometal conjugates in mitochondria.
[
99mTc(CO)3(9)] (white bars) and [99mTc(CO)3(10)] (black bar) without
additives, and [99mTc(CO)3(9)] in the presence of succinate (10 mM),
FCCP (0.5 μM), or sodium azide (20 mM).
Puzzled by the results obtained from these experiments we
resorted to investigating in more detail the binding of the radio-
conjugates to mitochondria. Subcellular fractionation following
internalization experiments did not reveal a statistically signifi-
cant accumulation of the dual-targeting conjugate [99mTc-
(CO)3(9)] in the target organelle (data not shown). To simplify
the experimental set-up, additional tests were conducted with
mitochondria isolated from PC-3 cells (see the Experimental
part). In brief, [99mTc(CO)3(L)] (L = 9, 10; 11.5 kBq and
15.3 kBq, respectively; 5 pmol of peptide) were incubated with
mitochondria (1 μg protein per mL) for 30–120 min (Fig. 6;
n = 4 for all experiments described henceforth). In these experi-
ments, the multifunctional conjugate [99mTc(CO)3(9)] exhibited
a steady and statistically significantly enhanced binding to mito-
chondria (10.15 1.41%) in comparison to reference compound
General procedures
Fmoc-amino acids, Rink Amide MBHA LL resin
(100–200 mesh), HOBt, HATU, and TBTU were purchased
from Merck Biosciences (Nottingham, UK). Bombesin (1–14)
trifluoroacetate and BocLysOMe were purchased from Bachem
(Bubendorf, Switzerland). Solvents and all other chemicals
were purchased from Acros Organics (Geel, Belgium), Merck
(Darmstadt, Germany), or Sigma Aldrich (Buchs, Switzerland)
and used as supplied unless stated otherwise. Polypropylene
syringes for manual peptide couplings, fitted with polypropylene
frits and a polypropylene plunger, were obtained from MultiSyn-
tech (Witten, Germany) and Teflon taps from Biotage (Uppsala,
Sweden). Na[99mTcO4] was eluted from
a Mallinckrodt
99Mo/99mTc generator (Tyco Healthcare, Petten, The Nether-
lands) using 0.9% saline. The precursor [99mTc(CO)3(H2O)3]+
was prepared by using the IsoLinkTM kit (Mallinckrodt-Tyco,
[
99mTc(CO)3(10)] (1.98 0.16%; unpaired t-test; p < 0.0001).
To the best of our knowledge these data represent the first report
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Petten, The Netherlands). Column chromatography was carried
out using silica gel C60 (particle size 0.04–0.063 mm) (Sigma)
and TLC was performed on precoated silica gel plates (0.25 mm,
degassed DMF (2–3 mL) were added to the resin. To the suspen-
sion was added tetrakis(acetonitrile)copper(I) hexafluorophos-
phate ([Cu(CH3CN)4]PF6) (0.015 mmol), tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine (TBTA) (0.015 mmol) and the
mixture was vigorously shaken at rt overnight. The resin was
washed with DMF and a solution of diethyldithiocarbamate
(0.5% in DMF) to remove the remaining copper species. The
completion of the reaction was verified by a colorimetric test for
solid-supported azides.43 The peptides were cleaved from the
resin and deprotected using a solution of trifluoroacetic acid,
phenol, water and triisopropylsilane (0.5–1 mL; 87.5/5/5/2.5%)
as a cleavage cocktail at rt for 2 h. After precipitation in ice-cold
diethyl ether (approx. 40 mL), the crude mixture of peptides was
recovered by centrifugation and washed twice with cold diethyl
ether. The precipitate was dissolved in water (5–10 mg mL−1),
purified by preparative HPLC, and lyophilized (Table 2).
1
60F254, Merck). H-, 13C- and 31P-NMR spectra were recorded
on a Bruker DPX 400 instrument. Chemical shifts are reported
in parts per million (ppm) relative to tetramethylsilane
(0.00 ppm) and coupling constants (J) in hertz (Hz). Standard
abbreviations indicating multiplicity are singlet (s), doublet (d),
doublet of doublets (dd), triplet (t), and multiplet (m). Analytical
and preparative HPLC were carried out with systems from Bis-
choff Chromatography, equipped with a λ-1010 UV/Vis and an
LB509 radioflow detector (Berthold Technologies), using C18
reversed-phase columns (Macherey Nagel Nucleodur C18 ISIS,
5 μm, 250 × 4.6 mm (column A) or Phenomenex Jupiter 4u
Proteo 90 Å, 4 μm, 250 × 4.6 mm (column B) for analysis and
Macherey Nagel Nucleodur C18 ISIS, 5 μm, 250 × 16 mm
(column C) or Nucleosil 100-5 C18, 5 μm, 250 × 21.0 mm
(column D) for purifications). HPLC solvents were 0.1% TFA in
H2O (A) and MeCN (B). Quality control of (radio)metal labeled
peptides was performed using column B and a linear gradient
from 80% to 30% A in 15 min (flow: 1.5 mL min−1); peptide
purification was performed using column C and a linear gradient
from 70% to 50% A in 15 min (flow: 8 mL min−1); purification
of compound 7 was done using column D and a linear gradient
from 55% to 35% A in 20 min (flow: 10 mL min−1). Log
D measurements were performed using column A and an
isocratic gradient 25% phosphate buffer (pH 7.4) and 75%
methanol (run time: 15–40 min; flow: 1 mL min−1). LRMS ana-
lyses were performed on a 4800 MALDI TOF/TOF™ analyzer
(Applied Biosystems) or on an ESI Bruker Esquire 3000 plus.
HRMS analyses were performed by LC-ESI on an LTQ Orbitrap
XL mass spectrometer (Thermo Scientific). Quantitative γ-count-
ing was performed on a COBRA II auto-gamma system (Model
5003; Packard Instruments).
In solution synthesis of building blocks and intermediates
Compounds 3 and 8 were prepared as previously described.14
Compound 6. The synthesis was accomplished according to a
modified procedure reported by Leavens et al.33 In brief, tris-
(p-methoxy)phenylphosphine (501 mg, 1.42 mmol) and 3-bromo-
propionic acid (363 mg, 2.37 mmol) in toluene (15 mL) were
stirred at reflux for 5 h and then cooled to rt. The mixture
was centrifuged, the precipitate was washed with toluene
(4 × 10 mL), and dried under reduced pressure. The crude
product was dissolved in acetonitrile (2 mL) and precipitated at
4 °C by addition of diethyl ether (12 mL). After drying under
reduced pressure, product 6 was isolated as a white solid
(bromide salt, 702 mg, 98%); purity according to HPLC > 95%;
1H NMR (MeOH-d4): δ = 7.68 (dd, 6H, JH–H = 9.0 Hz, JH–P
=
12.1 Hz), 7.27 (dd, 6H, JH–H = 9.0 Hz, JH–P = 2.6 Hz), 3.93 (s,
9H), 3.55–3.46 (m, 2H), 2.74–2.63 (m, 2H) ppm; 13C NMR
(MeOH-d4): δ = 173.7, 166.6 (d, JC–P = 3.0 Hz), 136.9 (d, JC–P
= 11.6 Hz), 117.3 (d, JC–P = 13.8), 110.3 (d, JC–P = 95.3 Hz),
56.6, 27.6 (d, JC–P = 52.5 Hz), 20.1 (d, JC–P = 57.8 Hz) ppm;
31P NMR (MeOH-d4): δ = 21.81 ppm (s); HRMS (ESI) m/z:
425.15119 [M]+ (calcd for C24H26O5P: 425.15124).
Solid phase synthesis
Automated solid-phase peptide synthesis (SPPS; scale:
0.1–0.25 mmol) was run on a Pioneer synthesizer (Applied Bio-
systems) using standard Fmoc chemistry with TBTU–HOBt as
coupling reagents and 20% piperidine in DMF as the deprotec-
tion reagent. The elongation was carried out using a 4-fold
excess of protected amino acids and coupling reagents. For
manual solid phase peptide synthesis, Fmoc-protected amino
acids or azidoacetic acid (3 equiv.) were coupled onto the resin
(scale: 0.03–0.1 mmol) in a syringe fitted with a polypropylene
frit and a Teflon tap in the presence of HATU (3 equiv.) and
DIPEA (5 equiv.) in DMF for 2 h. The completion of the reac-
tion was verified by the Kaiser test and repeated if necessary.
Elongation yields were determined by the UV-absorption of the
fluorenylmethylpiperidine adduct after treatment of the resin
with 5 mL of a 20% piperidine–DMF solution (3 × 3 min).
CuAAC on a solid support was carried out under argon in
oxygen-free solvents by a modified procedure reported for
CuAAC in solution.42 In brief, the resin (loaded with 0.03 mmol
peptide) was swollen several times in degassed DMF (5 mL).
The solvent was drained off thoroughly and a solution of lysine
derivatives 7 or 8 (0.06 mmol) and DIPEA (0.03 mmol) in
Compound 7. Compound
6 (bromide salt: 86.6 mg,
0.17 mmol) was dissolved under argon in CH2Cl2 (1.5 mL) and
SOCl2 (0.4 mL, 5.51 mmol) was added dropwise. The resulting
solution was stirred at rt overnight. The excess of SOCl2 was
removed with a nitrogen stream and then evaporated to dryness
under reduced pressure. The acid chloride of compound 6 was
dissolved under argon in CH2Cl2 (2 mL) at −78 °C, Et3N
(0.4 mL, 2.87 mmol) and compound 3 (34 mg, 0.12 mmol) were
added. The mixture was stirred for 4 h at −78 °C before quench-
ing the reaction by the addition of water (2 mL) followed by
warming to rt. The reaction mixture was washed with 0.1 M
citric acid and water. The aqueous phases were extracted twice
with CH2Cl2. The organic phases were combined, dried over
Na2SO4, and concentrated under reduced pressure. The Boc-pro-
tected intermediate was directly deprotected by stirring it in a
mixture of CH2Cl2–TFA (2 : 1; 6 mL) at rt for 5 h followed by
drying under reduced pressure. Purification by preparative HPLC
afforded compound 7 as a yellow oil (TFA salt; 50 mg; yield for
This journal is © The Royal Society of Chemistry 2012
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3 steps: 59%); purity according to HPLC >95%; 1H-NMR
(400 MHz, MeOH-d4; recorded after completed H/D exchange):
δ = 7.62 (dd, 6H, JH–H = 9.0 Hz, JH–P = 12.1 Hz), 7.21 (dd, 6H,
JH–H = 9.0 Hz, JH–P = 2.5 Hz), 4.04 (dd, 1H, JH–H = 6.6 Hz,
JH–H = 5.1 Hz), 3.99 (dd, 1H, JH–H = 16.7 Hz, JH–H = 2.6 Hz),
3.93 (dd, 1H, JH–H = 16.7 Hz, JH–H = 2.6 Hz), 3.88 (s, 9H),
3.49–3.38 (m, 2H), 3.16 (t, 1H, JH–H = 2.6 Hz), 3.06 (t, 2H,
JH–H = 6.6 Hz), 2.60–2.50 (m, 2H), 2.00–1.83 (m, 2H),
1.50–1.23 (m, 4H) ppm; 13C NMR (MeOH-d4): δ = 171.7
(d, JC–P = 13.6 Hz), 171.0, 166.6 (d, JC–P = 3.0 Hz), 136.9 (d,
Log D determination
Determination of log D values of radiotracers is usually accom-
plished by partitioning between n-octanol and PBS (pH 7.4)
using the “shake flask method”. However, the OECD guidelines
for testing of chemicals (Nr. 117) recommend an HPLC method
for the determination of log D values in the range of log
D 0–6.45 Applying this method also allows the evaluation of
radioactive and non-radioactive species, e.g., peptide precursors,
and their radioactive and non-radioactive metal-labeled
analogues. In brief, log D values of analytes were determined by
correlating their retention time with those of standard com-
pounds of known log D values using a calibration curve with at
least six reference compounds. All measurements were done
with a Bischoff HPLC system, column A and an isocratic mobile
phase composed of methanol (75%) and phosphate buffer
(pH 7.4; 25%) at a flow rate of 1 mL min−1. Standard com-
pounds (L-sodium ascorbate (t0), aniline (log D = 0.9), benzal-
dehyde (log D = 1.5), anisole (log D = 2.1), bromobenzene (log D
= 3.0), naphthalene (log D = 3.6), and dibenzyl (log D = 4.8);
0.1% in methanol)45 and testing compounds were injected three
times and analyzed either by UVor gamma detection. The differ-
ences in retention times (approx. 0.3 min) of radioactive analytes
JC–P = 11.6 Hz), 117.3 (d, JC–P = 13.8 Hz), 110.3 (d, JC–P
95.0 Hz), 79.7, 74.4, 60.1, 56.6, 55.0, 40.3, 36.5, 30.0 (d, JC–P
=
=
7.2 Hz), 29.2 (d, JC–P = 2.8 Hz), 23.2, 20.2 (d, JC–P = 57.8 Hz)
ppm; 31P NMR (MeOH-d4): δ = 21.92 ppm (s); HRMS (ESI)
m/z: 591.26208 [M]+ (calcd for C33H40O6N2P: 591.26185).
(Radio)metal labeling of peptides
Na[99mTcO4] was eluted from a Mallinckrodt 99Mo/99mTc
generator (Tyco Healthcare, Petten, The Netherlands) using
0.9% NaCl. The precursor [99mTc(CO)3(H2O)3]+ was prepared
according to published procedures.44 In brief, [99mTcO4]− (1 mL,
1.5–2 GBq) in 0.9% NaCl was added to the IsoLinkTM kit
(Mallinckrodt-Tyco, Petten, The Netherlands) and heated for
20 min at 100 °C. After the solution was cooled to rt, the pH
was adjusted to pH 7 with a 1 : 2 mixture of 1 M phosphate
buffer (pH 7.2) and 1 M HCl (final pH 1.4). Aliquots of 1 mM
stock solutions of the peptides 9 or 10 in water (10–20 μL;
10–20 nmol) were added to a solution of [99mTc(CO)3(H2O)3]+
(90–180 μL; 100–250 MBq) to reach a final peptide concen-
tration of 0.1 mM. The reaction mixture was heated at 100 °C
for 30 min and the radiochemical yield and purity of [99mTc-
(CO)3(L)] (L = 9, 10) was determined by HPLC.
(
99mTc-complexes) and the corresponding non-radioactive Re-
compounds as a result of the serial arrangement of the UV/Vis
and γ-detectors were corrected by analysis of co-injections. The
calibration curve was calculated by linear regression analysis
using GraphPad Prism 5.0 (see ESI†).
In vitro experiments
Cell culturing. Human Caucasian prostate adenocarcinoma
(PC-3) cells were obtained from HPA Culture Collections
(Salisbury, UK) and cultured at 37 °C and 5% CO2 in
Dulbecco’s modified Eagle’s medium (DMEM, high glucose)
containing 10% (v/v) heat-inactivated fetal bovine serum (FBS
superior, OXOID, Pratteln, Switzerland), L-glutamine (200 mM),
100 IU mL−1 penicillin and 100 μg mL−1 streptomycin. All
culture reagents, except FBS, were purchased from BioConcept
(Allschwil, Switzerland). The cells were subcultured weekly
after detaching them with a commercial solution of trypsin–
EDTA (1 : 250) in PBS.
Rhenium complexes were prepared by mixing aqueous stock
solutions of peptides 9 or 10 (100 μL, 1 mM) with [Re(CO)3Br3]-
34
[N(Et)4]2 (150 μL, 1 mM) and heating at 100 °C for 60 min.
The products were purified by HPLC and analyzed by MS.
[Re(CO)3(9)]: quantitative conversion of peptide 9 (HPLC);
purity >99%; MS (MALDI TOF) m/z: 2078.8 [M]+ (calcd for
C91H122N21O22PRe: 2078.84). [Re(CO)3(10)]: quantitative con-
version of peptide 10 (HPLC); purity >99%; MS (MALDI TOF)
m/z: 1714.7 [M + H]+ (calcd for C69H100N21O19Re: 1713.71).
Saturation binding assay. On the day prior to the experiment
PC-3 cells (106 cells per well) were plated in a six-well plate and
incubated at 37 °C and 5% CO2 overnight, allowing them to
attach. The next day, the cell-culture medium (1% FBS) was
changed (0.8 mL) and the cells were kept in the fridge for
30 min to stop cell activity prior to the start of the experiment. In
order to reach receptor saturation, the cells were incubated with
increasing concentrations (0.1, 0.5, 1, 5, 10, 20, 50, and 100 nM
per well; each 100 μL) of the radiolabeled peptides [99mTc-
(CO)3(L)] (L = 9, 10). Non-specific receptor binding was deter-
mined using an excess of natural bombesin (2.5 μM per well for
concentrations of the radiopeptide <10 nM, and 10 μM per well
for higher concentrations). After incubation at 4 °C for 2 h, the
supernatant was removed and the cells were washed twice with
1 mL of ice-cold PBS (pH 7.4). The combined supernatants rep-
resent the free, unbound radiopeptide fraction. To determine the
Stability studies
The radiolabeled peptides [99mTc(CO)3(L)] (L = 9, 10) (100 μL;
100 pmol, 0.6 MBq) were incubated at 37 °C with 900 μL PBS
(pH 7.4), NaCl (0.9%), or a cell culture medium (1% FBS)
respectively. At different time points (1, 2, and 4 h), aliquots
were analyzed by HPLC (n = 3). In the case of the cell culture
medium, serum proteins were precipitated prior to HPLC analy-
sis by the addition of aliquots to ethanol followed by centrifu-
gation (2500g, 5 min).
Histidine challenge. Compounds [99mTc(CO)3(L)] (L = 9, 10)
(100 μL; 200 pmol, 1.1 MBq) were incubated at 37 °C with
900 μL of a solution of histidine (1 mM in PBS; 4500-fold
excess). Aliquots were analyzed by HPLC after 1, 4, and 24 h
(n = 3).
7600 | Org. Biomol. Chem., 2012, 10, 7594–7602
This journal is © The Royal Society of Chemistry 2012

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Mitochondria-binding experiments. Mitochondria were iso-
lated from cultured PC-3 cells using the Thermo SCIENTIFIC
mitochondria isolation kit for cultured cells according to the pro-
cedure described in the operating manual. Isolated mitochondria
obtained by this procedure maintain their integrity.46,47 Protein
concentration was measured by the Bradford method using the
Bio-Rad protein assay and BSA as a standard. Mitochondria
binding experiments were performed following procedures pub-
lished by Murphy et al.38,39 In brief, isolated mitochondria were
resuspended in KCl medium (120 mM KCl, 10 mM Hepes
(pH 7.2), and 1 mM EGTA). The mitochondria (0.9 mL, 1 μg
protein per mL) were incubated with 100 μL of each radiolabeled
peptide [99mTc(CO)3(L)] (L = 9, 10; 5 pmol; 11.5 and 15.3 kBq,
respectively) at rt on a shaking plate for 30 min. To investigate
the specificity of [99mTc(CO)3(9)], mitochondria were preincu-
bated with sodium succinate (10 mM), FCCP (0.5 μM) or
sodium azide (20 mM)40 respectively for 10–30 min prior to the
addition of the radiolabeled conjugate. After the experiments,
the mitochondria were pelleted (centrifugation at 12 000g for
5 min) and the supernatant was removed. The pellet was washed
with KCl medium (1 mL), the supernatants combined and
measured in a gamma counter; this fraction represents unbound
radioactivity. The mitochondria precipitate was resuspended in
1 mL of triton X-100 (10% v/v) and measured in a gamma
counter to quantify the amount of the radioactive peptide associ-
ated with the mitochondria relative to total added activity (n = 4
for all experiments).
receptor bound fraction, the cells were lysed with 1 M NaOH
(1 mL) for 10 min at 37 °C and the wells were washed twice
with 1 M NaOH (1 mL). The free and receptor bound fractions
were measured in a gamma counter for quantification. Dis-
sociation constants (Kd) were calculated from the data for
specific binding with nonlinear regression using GraphPad
Prism5, normalized to 106 cells (n = 3 in triplicate).
Internalization studies. On the day prior to the experiment,
PC-3 cells (106 cells per well) were placed in six-well plates
with a cell culture medium (1% FBS) and incubated at 37 °C,
5% CO2 overnight to let them attach. On the day of the experi-
ment, the medium was removed, and cells were incubated with
fresh medium (1.3 mL) for 1 h. Radiolabeled peptides [99mTc-
(CO)3(L)] (L = 9, 10; per well: 100 μL; 0.25 pmol; 1.5 kBq)
were added and the cells were incubated for different time points
(10, 20, 30, 60, 90, and 120 min) in triplicate to allow binding
and internalization. Non-specific receptor binding and internaliz-
ation was determined in the presence of a 1000-fold excess of
natural bombesin (250 pmol; 100 μL per well) as the receptor
blocking agent. After each time point, the supernatant was col-
lected, and the cells were washed twice with PBS (1 mL;
pH 7.4). The combined supernatants represent the free, unbound
radiopeptide fraction. Receptor-bound radioactivity was isolated
by incubating the cells twice for 5 min with an acidic glycine
solution (100 mM NaCl, 50 mM glycine, pH 2.8; 1 mL) on ice
followed by removal of the supernatant. The internalized fraction
was determined by cell lysis with 1 M NaOH (1 mL; 10 min;
37 °C, 5% CO2) and the wells containing the cell lysate were
washed twice with NaOH (1 M, 1 mL). All fractions were
measured radiometrically in a gamma counter and calculated as
percentage of applied dose normalized to 106 cells (n = 3 in
triplicate).
Conclusion
In summary, we report the synthesis and in vitro evaluation of
novel dual-targeting radiopeptide conjugates with the goal of
improving the cellular retention of radioactivity in cancer cells
after specific delivery. Starting from readily accessible building
blocks, a tumor-targeting peptide ([Nle14]BBS(7–14)), a 99mTc-
based SPECT reporter probe ([99mTc(CO)3]+), and a mitochon-
dria-seeking triphenylphosphonium (TPP) moiety were selec-
tively and efficiently assembled into a trifunctional conjugate.
With the exception of the radiometal labeling step, the assembly
of the described multifunctional conjugates was accomplished
conveniently on a solid support. While the first generation conju-
gate bound and internalized specifically into GRP receptor
expressing PC-3 cells, it did not display specific accumulation in
its intracellular target, the mitochondria, resulting in an overall
unaltered externalization rate of radioactivity from PC-3 cells.
Nevertheless, the combination of two different entities specific
for an extra- and an intracellular target in the same radioconju-
gate is an attractive approach to improve the efficacy of radio-
tracers. We are currently employing the same modular synthetic
strategy for the development of a simplified, second generation
of dual-targeting radiopeptide conjugates in an effort to identify
the features required for improving the retention time of radio-
activity in cancer cells and tumors.
Externalization studies. PC-3 cells (106 cells per well) were
placed in six-well plates with a cell culture medium (1% FBS)
and incubated at 37 °C and 5% CO2 overnight to let them attach.
On the day of the experiment, the medium was removed and
cells were incubated with fresh medium (1.3 mL) for 1 h. Radio-
labeled peptides [99mTc(CO)3(L)] (L = 9, 10; per well: 100 μL;
0.25 pmol; 1.3 kBq or 2.5 pmol; 13.7 kBq) were added and the
cells incubated for 1 h in triplicate to allow internalization into
the cells. Non-specific receptor binding and internalization was
determined in the presence of a 1000-fold excess of natural bom-
besin (250 pmol or 2500 pmol; 100 μL per well) as the receptor
blocking agent. After 1 h, the supernatant was collected (free
fraction) and receptor-bound radiopeptides were removed by
washing the cells two times for 5 min with an acidic glycine
buffer on ice (bound fraction). A fresh cell culture medium
(1 mL) was added and the cells were incubated for 10, 20, 30,
60, 90, 120, 150, 180, 240, and 300 min. At each time-point, the
supernatant was collected and cells were washed twice (1 mL
PBS; pH 7.4). These fractions represent the externalized amount
of radioactivity. The remaining internalized amount of radio-
activity was recovered by cell lysis as described above. All frac-
tions were measured in a gamma counter for quantification
calculated as percentage of the total internalized fraction at
t = 0 min (n = 3 in triplicate).
Acknowledgements
We thank Covidien for providing IsolinkTM kits, and Kayhan
Akyel, Dieter Staab, Christian Guenat and Ingo Muckenschnabel
This journal is © The Royal Society of Chemistry 2012
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