Orthogonally protected artificial amino acid as tripod ligand for automated peptide synthesis and labeling with [99mTc(OH2) 3(CO)3]+

Article abstract of DOI:10.1021/bc3003327

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [ ^99mTc(CO)₃]⁺ core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH₂ group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH₂ group of the α-amino acid portion is Fmoc- and the -NH₂ of Dap are Boc-protected. Fmoc-l-Lys(Dap(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7-14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys(Dap). For all peptides, quantitative labeling with the [^99mTc(CO)₃]⁺ core at a 10 μM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re(OH₂)₃(CO) ₃]⁺ and Lys(Dap)-BBN(7-14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC₅₀ of cyclo-(RGDyK(Dap[Re(CO)₃])) for α_vβ₃ is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α _vβ₃-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield ^99mTc-labeling with [^99mTc(OH₂) ₃(CO)₃]⁺, forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.

Full text of DOI:10.1021/bc3003327

Article
pubs.acs.org/bc
Orthogonally Protected Artificial Amino Acid as Tripod Ligand for
Automated Peptide Synthesis and Labeling with [99mTc(OH2)3(CO)3]+
Yunjun Shen,† Margret Schottelius,‡ Karel Zelenka,† Mariarosaria De Simone,§,∥ Karolin Pohle,‡
Horst Kessler,§,∥ Hans-Jurgen Wester,‡ Paul Schmutz,† and Roger Alberto*,†
̈
†Institute of Inorganic Chemistry, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland
̈
̈
‡Lehrstuhl fur Pharmazeutische Radiochemie, TU Munchen, Walther-Meißner-Str. 3, D-85748 Garching, Germany
̈
̈
§Institute for Advanced Study at the Department Chemie, TU Munchen, Lichtenbergstraße 4, D-85748 Garching, Germany
∥Chemistry Department, King Abdulaziz University, Saudi Arabia
̈
S
* Supporting Information
ABSTRACT: 1,2-Diamino-propionic acid (Dap) is a very
strong chelator for the [99mTc(CO)3]+ core, yielding small and
hydrophilic complexes. We prepared the lysine based Dap
derivative L-Lys(Dap) in which the ε-NH2 group was replaced
by the tripod through conjugation to its α-carbon. The
synthetic strategy produced an orthogonally protected bifunc-
tional chelator (BFC). The -NH2 group of the α-amino acid
portion is Fmoc- and the -NH2 of Dap are Boc-protected. Fmoc-L-Lys(Dap(Boc)) was either conjugated to the N- and C-
terminus of bombesin BBN(7−14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced
the native lysine in a cyclic RGD peptide with L-Lys(Dap). For all peptides, quantitative labeling with the [99mTc(CO)3]+ core at
a 10 μM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from
[Re(OH2)3(CO)3]+ and Lys(Dap)-BBN(7−14) or cyclo-(RGDyK(Dap)), respectively. Determination of integrin receptor
binding showed low to medium nanomolar affinities for various receptor subtypes. The IC50 of cyclo-(RGDyK(Dap[Re(CO)3]))
for αvβ3 is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude
mice showed reasonable αvβ3-integrin specific tumor uptake. Altogether, orthogonally protected L-Lys(Dap) represents a highly
versatile building block for integration in any peptide sequence. Lys(Dap)-precursors allow high-yield 99mTc-labeling with
[
99mTc(OH2)3(CO)3]+, forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with
excellent in vivo characteristics.
labeling method chosen. For example, the use of radiometals
requires the functionalization of the biomolecule with an
usually bulky chelator for radiometal complexation. Even if this
modification is well-tolerated in a given position of the peptide
sequence and does not seriously challenge receptor affinity, it
may still have substantial impact on the overall pharmacoki-
netics of the peptide radiopharmaceutical. This problem has
been repeatedly observed for 99mTc-labeled peptides, and
recent research has therefore been focused on the development
of suitable bifunctional chelators (BFC) allowing 99mTc-labeling
with high efficiency, leading to complexes with high stability.
The influence of the type of chelator on labeling efficiencies
and complex stabilities has recently been reviewed compre-
hensively several times.8−12 However, in some cases, coordina-
tion chemistry and physicochemical characteristics such as
lipophilicity of the complexes were not optimally compatible
with the requirements for in vivo imaging applications.
INTRODUCTION
■
Peptide-based targeting vectors as carriers for radionuclides are
the focus of finding and developing new radiolabeled
compounds for molecular imaging purposes. Since distinct
peptide receptors are strongly overexpressed in many tumor
cells; they represent attractive targets for radionuclide-based
diagnosis and therapy in oncology.1−7 Most peptide radiophar-
maceuticals, which are either used in clinical studies or
currently under preclinical evaluation, are structurally derived
from naturally occurring peptide ligands. These have intrinsi-
cally evolved to be highly active ligands, but also, being mostly
peptide hormones, to be rapidly degraded in vivo.2 For the
applicability of a radiolabeled peptide probe for in vivo imaging,
however, high metabolic stability, suitable pharmacokinetics
(e.g., fast clearance from the blood pool, low binding to plasma
proteins or nontarget tissues, fast renal excretion) and high
receptor affinity are crucial. Even if all these issues have been
carefully addressed and an “optimized” small and metabolically
stable peptide sequence with high affinity toward the target
receptor is available, introduction of the radiolabel may
represent a serious challenge on both receptor affinity and in
vivo pharmacokinetics, depending on the radionuclide and
Received: June 21, 2012
Revised: November 22, 2012
Published: December 14, 2012
© 2012 American Chemical Society
26
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Bioconjugate Chemistry
Article
a
Scheme 1. Basic Bifunctional, L-Lysine Based Chelators
a
1,2-Diamino-propionic acid (Dap) as chelating unit conjugated to the ε-position (1),26,27 the corresponding, orthogonally protected BFC (2) for
automated SPPS, the LAT1 targeting radiopharmaceutical [99mTc(1)(CO)3], and the bifunctional SAAC system.16−18
As a labeling precursor, the complex [99mTc(OH2)3(CO)3]+
has attracted much attention over the past years. The
{
chemical shifts are in ppm; coupling constants (J) are given in
Hz. High-resolution electrospray ionization mass spectrometry
was performed on a FinniganMAT 900 (Finnigan MAT95, San
Jose, CA; USA) double-focusing magnetic sector mass
spectrometer (geometry BE). Compounds 3, 4, 5, and 6
were prepared according to literature. Characterization data
(1H and 13C NMR, MS) were in agreement with literature
procedures.30,31
For the preparation of the peptides L1−L4 coordinated to
the {Re(CO)3}+ moiety, we used (NEt4)2[ReBr3(CO)3] as
starting material as described in the experimental procedures.
Once dissolved in water, [ReBr3(CO)3]2− converts rapidly to
[Re(OH2)3(CO)3]+ which then coordinates to the correspond-
ing ligand.
( S ) - B e n z y l 2 - ( b e n z y l o x y c a r b o ny l a m i n o ) - 6 -
(methylsulfonyloxy)hexanoate (7). To a stirred solution of 6
(2g, 5.4 mmol) and H3CSO2Cl (0.466 mL, 6 mmol) in CH2Cl2
(10 mL) cooled to −78 °C, NEt3 (0.797 mL, 6 mmol) was
slowly added. The mixture was allowed to come to r.t., and
stirring was continued for 3 h. The solution was diluted with 10
mL CH2Cl2, washed with H2O, dried over MgSO4, and
evaporated. The crude product was purified by flash column
chromatography (33% Et2O in hexane) to give 7 as a pale
yellow oil (2.3g, 5.12 mmol, 95%). Rf = 0.77 (SiO2, TLC,
Et2O). 1H NMR (400 MHz, CDCl3): δ 7.26 (s, 10H), 5.61 (d,
1H, J = 8 Hz), 5.02−5.13 (m, 4H), 4.34 (d, 1H, J = 5.2 Hz),
4.03 (t, 2H, J = 6.2 Hz), 2.81 (s, 3H), 1.32−1.78 (m, 6H). Anal.
Calcd. for C22H27NO7S: C, 58.78; H, 6.05; N, 3.12. Found: C,
58.39; H, 5.99; N, 3.04. MS-ESI: m/z 472.1 (calcd. 472.15)
[(M + Na)+].
(7S)-8-Benzyl 1-ethyl 2-(tert-butoxycarbonylamino)-7-
(benzyloxycarbonylamino)-2-cyan ooctanedioate (8). Na°
(0.046g, 2 mmol) was dissolved in abs. ethanol, and ethyl 2-
(tert-butoxycarbonylamino)-2-cyanoacetate (0.46g, 2 mmol)
was added; the mixture was stirred at 60 °C for 30 min. After
cooling to r.t., ethanol was removed under vacuum, the residue
dissolved in DMSO (5 mL), mesylate 7 (0.67g, 1.5 mmol) was
added, and the resulting solution stirred at 70 °C for 10 h.
DMSO was removed under high vacuum and the residue
dissolved in EtOAc. The solution was washed with water, brine,
dried over MgSO4, and concentrated in vacuo. Purification on a
silica gel column with EtOAc/hexane provided 8 (0.66g, 76%)
as a colorless oil. Rf = 0.24 (SiO2, TLC; hexane/EtOAc, 2/1).
1H NMR (400 MHz, CDCl3): δ 7.26 (s, 10H), 5.41 (d, 1H, J =
7.6 Hz), 5.04−5.11 (m, 4H), 4.33 (s, 1H), 4.22 (t, 2H, J = 7
Hz), 1.58−1.96 (m, 4H), 1.38 (s, 9H), 1.18−1.31 (m, 7H).
99mTc(CO)3}+ core accepts a wide variety of different ligands
due to the robust nature of the resulting complexes.13−15
Among these BFCs, Valliant et al. introduced the L-lysine-based
single amino acid chelator (SAAC) approach. In this concept, a
BFC is obtained by modifying the ε-amino group with a
heterocyclic tridentate, originally pyridine-based tripod.16−18
Tether and ligand are altered while the entire ligand can be
subjected to automated SPPS. The utility of this concept was
demonstrated by the labeling of many peptides prepared via
this approach.11,19−24 A drawback of the concept is the
lipophilicity of the tagged complex and its relatively large
size. Lipophilicity can be reduced by ligand variations, however,
but at the cost of molecular weight and label size.25
We have introduced a BFC (1) based on L-lysine in which
the ε-amino group was replaced by 2,3-diamino propionic acid
(Dap). Labeling of 1 with the {99mTc(CO)3}+ fragment
resulted in [99mTc(1)(CO)3] which was recognized and actively
transported by the L-type amino acid transporter LAT1,
demonstrating for the first time that small molecules can be
labeled with 99mTc under retention of transporter and receptor
affinity.26 The α-amino group allows introduction of further
functionalities for conjugating a wide variety of biomolecules
with the small, hydrophilic, and strong Dap chelator.27−29 Since
[
99mTc(1)(CO)3] basically represents an “unnatural amino
acid”, the use of appropriately and orthogonally protected
precursors should allow the substitution of structurally similar
amino acids in a peptide sequence during peptide synthesis
without strongly affecting receptor affinities.
We present in this work the synthesis of the bifunctional
artificial amino acid 2. BFC 2 contains orthogonally protected
functional groups which enable its general use in automated
SPPS. Exemplary incorporation into BBN(7−14) and an RGD
peptide demonstrate the strength of this concept (Scheme 1).
The resulting peptides were labeled with the {99mTc(CO)3}+
and {Re(CO)3}+ cores and evaluated in vitro and in vivo.
EXPERIMENTAL SECTION
■
General. All reagents and organic solvents were purchased
from Aldrich, Acros, or ABCR in reagent grade or better and
used without further purification. Column chromatography was
accomplished on silica gel 60 (20−40 mesh). Electrospray
ionization mass spectrometry (ESI-MS) spectra were recorded
a Bruker HCT spectrometer and NMR spectra on a Bruker 400
or 500 MHz spectrometer in CDCl3 solutions. All NMR
27
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Article
Anal. Calcd. for C31H39N3O8: C, 64.01; H, 6.76; N, 7.22.
Found: C, 63.92; H, 6.68; N, 7.18. MS-ESI: m/z 582.4 (calcd.
582.27) [(M + H)+].
protected amino acids with appropriate side-chain protection
were used for the synthesis. The final products were cleaved
from the resin by a standard procedure using a cocktail
containing thioanisol, water, phenol, and trifluoroacetic acid in
a ratio of 5:5:5:85 and precipitated into methyl t-butyl ether. At
this point, the carboxylate group in the Dap unit was still
esterified. Coordination to the {Re(CO)3}+ or to the
(7S)-8-Benzyl 1-ethyl 2-(tert-butoxycarbonylamino)-7-
(benzyloxycarbonylamino)-2-((tert-butoxycarbonylamino)-
methyl)octanedioate (9). Boc2O (0.87g, 4 mmol) was added
to a solution of 8 (1.20g, 2 mmol) in dry MeOH of 0 °C
followed by NiCl2·6H2O (0.048g, 0.2 mmol). NaBH4 (0.606g,
16 mmol) was then added in small portions over 30 min. The
resulting mixture was allowed to warm to r.t. and left to stir for
a further 2 h. The mixture was filtered through Celite and the
filtrate evaporated to dryness in vacuo. The residue was taken
up in EtOAc, washed with water, brine, dried over MgSO4, and
concentrated. Flash chromatography on silica gel column
eluting with EtOAc/hexane provided 9 (0.96g, 70%) as a
{
99mTc(CO)3}+ moiety hydrolyzes this group concomitantly.
Crude peptides were purified by preparative HPLC (gradient
from 0% to 100% solvent B over the course of 60 min). ESI-MS
was used to determine the molecular constitution of the
conjugates. ESI-MS for L1, L2, L3: m/z 1183.7 [(M + H)+].
Synthesis of RGD-Peptide Cyclo-(Arg-Gly-Asp-D-Tyr-Lys-
(Dap)) (L4). The peptide was synthesized by solid-phase
peptide synthesis (SPPS) on 2-chlorotrityl chloride resin using
Fmoc-strategy. The resin was loaded with Fmoc-Gly-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Dap)-OH, Fmoc-D-Tyr(tBu)-
OH, and Fmoc-Asp(OtBu) were sequentially coupled with
HBTU (4 equiv), HOBt (4 equiv), and DIEA (10 equiv) in
DMF. Cleavage of protected linear peptide 11 from the resin
was performed without affecting the side-chain protecting
groups by using mildly acidic conditions (0.8% TFA in DCM
or HOAc/TFA/DCM = 1:1:8). Head-to-tail cyclization to
afford compound 12 was carried out in DMF with HBTU (4
equiv), HOBt (4 equiv), and DIEA (10 equiv). Finally, the
ethyl ester was hydrolyzed with LiOH in a mixture of MeOH
and H2O and the remaining side-chain protecting groups were
removed by treatment with trifluoroacetic acid/water/triiso-
propylsilane (TIPS) (95:2.5:2.5) at room temperature for 1.5 h
to yield L4. The reaction mixture was concentrated and
precipitated with ice-cold ether and purified by preparative
HPLC (gradient from 5% to 25% solvent B over the course of
70 min). ESI-MS: m/z 707.3 [(M + H)+], 354.1 [(M + 2H)2+].
Synthesis of [Re(L4)(CO)3] (13), [Re(L1)(CO)3] (14), [Re-
(L2)(CO)3] (15), and [Re(L3)(CO)3] (16). An excess of
(NEt4)2[ReBr3(CO)3] was added to 6 mg of an aqueous
solution of L1, L2, L3, or L4, respectively. The solution was
heated at 70 °C for 6−20 h (longer times do not reduce the
yields) with stirring. Quality control of the reaction mixture was
done by RP-HPLC. The desired product was purified by
HPLC, lyophilized, and characterized by ESI-MS. ESI-MS for
14, 15, 16: m/z 1425.6 [(M + H)+], 713.3 [(M + 2H)2+], 13:
m/z 977.3 [(M + H)+].
Determination of Binding Affinity and Specificity
Using Solubilized Integrins. The inhibitory activity and
selectivity of L4 and [Re(L4)(CO)3] were determined using an
ELISA assay based on previously reported methods with some
optimizations.32,33
Human integrins αvβ3 and αvβ5 were purchased from
Millipore, αIIbβ3 from Enzyme Research Laboratories, and
α5β1 and αvβ3 from R&D Systems. Vitronectin was purchased
from Millipore, fibronectin from Sigma, fibrinogen from
Calbiochem, and LAP protein from R&D Systems.
Blocking and binding steps were always performed with TS
buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2,
1 mM MgCl2, and 1 mM MnCl2) containing 1% BSA (TSB-
buffer). Washing steps after the incubation time were done with
PBST buffer (10 mM Na2HPO4, pH 7.5, 150 mM NaCl, and
0.01% Tween 20).
For the integrins αvβ3 and αvβ5, the binding was visualized
using a mouse antihuman integrin α-V monoclonal antibody for
αv subunit (MAB 1978 purchased from Chemicon); for α5β1
and αIIbβ3 were used antibodies from BD Biosciences (mouse
1
colorless oil. Rf = 0.4 (SiO2, TLC; hexane/EtOAc, 2/1). H
NMR (500 MHz, CDCl3): δ 7.24 (s, 10H), 5.42 (s, 1H), 5.35
(d, 1H, J = 7.5 Hz), 5.04−5.11 (m, 4H), 4.82 (s, 1H), 4.31 (s,
1H), 4.11 (d, 2H, J = 6.5 Hz), 3.60 (m, 2H), 1.57−1.99 (m,
4H), 1.41 (s, 18H), 1.06−1.20 (m, 7H). Anal. Calcd. for
C36H51N3O10: C, 63.05; H, 7.50; N, 6.13. Found: C, 62.90; H,
7.23; N, 6.00. MS-ESI: m/z 708.5 (calcd. 708.35) [(M + Na)+].
(2S)-2-Amino-7-(tert-butoxycarbonylamino)-7-((tert-
butoxycarbonylamino)methyl)-8-ethoxy-8-oxooctanoic acid
(10). Thirty milligrams of 10% Pd/C was added to an
anhydrous EtOH (15 mL) solution of 9 (0.27g, 0.4 mmol).
H2 was then bubbled through the solution for 2 days at r.t. The
solution was filtered with Celite to remove the catalyst and the
solvent removed by rotary evaporation. The residue was
purified by C18 RP chromatography (1:1 CH3OH/H2O) to
1
give 10 (0.16 g, 88%) as a white solid. H NMR (400 MHz,
CDCl3): 7.94 (s, 1H), 5.73 (s, 1H), 5.15 (s, 1H), 4.12 (d, 1H, J
= 6 Hz), 3.57 (m, 3H), 1.41 (s, 18H), 1.18−1.82 (m, 11H).
MS-ESI: m/z 484.4 (calcd. 484.27) [(M + Na)+]. HRMS
(ESI): Calcd for [M+H]+, 462.2815; Found, 462.2804.
(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-7-(tert-
butoxycarbonylamino)-7-((tert-butoxycarbonylamino)-
methyl)-8-ethoxy-8-oxooctanoic acid (2). Compound 10
(0.6g, 1.3 mmol) was dissolved in dioxane (4 mL) and a 5%
aqueous solution of Na2CO3 (8 mL). The reaction mixture was
immersed in an ice bath and a solution of FmocCl (0.336g, 1.3
mmol) in dioxane(4 mL) was added. The solution was allowed
to warm to r.t. and stirred for 3 h. The solvent was evaporated
under high vacuum and the resulting material was purified on a
silica gel column eluting with CHCl2/MeOH. Compound 2
was received as a white solid (0.77g, 87%). Rf =0.3 (SiO2, TLC;
CH2Cl2/MeOH, 30:1). 1H NMR (400 MHz, CDCl3): δ 7.26−
7.77 (m, 8H), 5.53 (s, 1H), 4.89 (s, 1H), 4.19−4.50 (m, 5H),
3.68 (m, 2H), 1.41 (s, 18H), 1.25−2.10 (m, 11H). MS-ESI: m/
z 682.5 (calcd. 682.34) [(M−H)−]. HRMS (ESI): Calcd for
[M+Na]+, 706.3315; Found 706.3314.
Synthesis of BBN Analogues (L1−L3). Peptides were
prepared either on an automated peptide synthesizer or by
manual synthesis using Fmoc chemistry. The BBN analogues
prepared and characterized were as follows: NH2-Lys(Dap)-
Gln-Trp-Ala-Val-Gly-His-Leu-Met-CONH2 (N-terminal, L1),
NH2-Gln-Trp-Ala-Val-Lys(Dap)-Gly-His-Leu-Met-CONH2
(integrated, L2), and NH2-Gln-Trp-Ala-Val-Gly-His-Leu-Met-
Lys(Dap)-CONH2 (C-terminal, L3). Peptide synthesis used
traditional Fmoc chemistry with HBTU or HATU activation of
the carboxylate groups on the reactant for coupling with the N-
terminal amino group on the growing peptide anchored via the
C-terminus to the resin. Rink amide MBHA resin and Fmoc-
28
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antihuman CD49e for α5β1 and mouse antihuman CD41b for
αIIbβ3) and Sigma (antimouse IgG-peroxidase). Peroxidase
development was performed using the substrate solution
3,3,5,5′-tetramethylethylenediamine (TMB from Seramun
Diagnostic GmbH) and 3 M H2SO4 to stop the reaction.
The absorbance (450, 492 nm) was recorded with a
POLARstar Galaxy plate reader (BMG Labtechnologies).
Every concentration was analyzed in duplicate and the resulting
inhibition curves were analyzed using OriginPro 7.5G software.
Each plate also contained either Tirofiban34 or Cilengitide35 as
reference compounds.
αvβ3 Assay. Flat-bottom 96-well ELISA plates (from Brand)
were coated with 100 μL of vitronectin (2 μg/mL) overnight at
4 °C in 15 mM of Na2CO3, 35 mM NaHCO3, pH 9.6
(carbonate buffer).
After removal of the coating solution, the wells were blocked
for 1 h at r.t. with 150 μL/well of TSB. Plates were then washed
three times with 200 μL/well of PBST.
2.0 μg/mL (1:250 dilution) and secondary antibody (anti-
mouse IgG-peroxidase) at 1.0 μg/mL (1:770 dilution) for 1 h
at r.t. All subsequent steps were performed as described for
αvβ3.
Determination of Binding Affinity Using M21 Cells.
The in vitro integrin binding affinities were assessed via a
cellular displacement assay using 125I-echistatin (Perkin-Elmer,
Rodgau, Germany; specific activity: 2200 Ci/mmol) as an
integrin specific radioligand on M21 human melanoma cells.
One day prior to the experiment, M21 cells were harvested
using trypsin/EDTA (0.05% and 0.02%) in PBS (Biochrom),
centrifuged, and resuspended in culture medium. The cells
were transferred into 24-well plates (approximately 200 000
cells per well in 1 mL) and placed in the incubator overnight.
The plates were used when confluence reached approximately
80%. Before the experiment, the culture medium was removed
and binding buffer (20 mM tris(hydroxymethyl)aminomethane
(Tris) pH 7.4, 150 mM NaCl, 2 mM CaCl2·2H2O, 1 mM
MgCl2·6H2O, 1 mM MnCl2·4H2O, 0.1% (m/m) BSA) was
added. The cells were incubated with 125I-echistatin (120 000−
140 000 cpm/well) and increasing concentrations of the RGD
ligands (10−11−10−4 mol/L), both dissolved in binding buffer.
The total incubation volume was adjusted to 500 μL by
addition of binding buffer. After the cells were incubated for 2 h
at r.t., the supernatant was removed and the cells were washed
twice with cold PBS. The cells were lysed with 1 M sodium
hydroxide solution and transferred to vials. Quantification of
bound radioactivity was performed using a gamma counter. The
experiments were performed in duplicate and repeated three
times. The best-fit IC50 (inhibitory concentration of 50%)
values were calculated by fitting the data by nonlinear
regression using GraphPad Prism (GraphPad Software, Inc.).
Next, 4.0 μg/mL of soluble integrin αvβ3 and a serial dilution
of integrin inhibitors and the control Cilengitide were
incubated in the coated wells for 2 h at r.t.
After washing three times, the plate was treated with 100 μL/
well of primary antibody (MAB1978 diluted 1:500 in TSB) and
the secondary antibody (antimouse IgG-peroxidase) at 2.0 μg/
mL for 1 h at r.t. After three washing steps, integrin binding was
visualized with TMB. The oxidation was allowed to proceed for
5 min, and the product absorbance was measured at 450 nm.
αvβ6 Assay. For this assay, ELISA plates were coated with
100 μL of LAP protein (0.4 μg/mL) overnight at 4 °C in
coating TS buffer. Blocking and washing steps were performed
as described for αvβ3. Next, 0.5 μg/mL of soluble integrin αvβ6
and a serial dilution of integrin inhibitors and the control
Cilengitide were incubated in the coated wells for 1 h at r.t. All
subsequent steps were performed as described for αvβ3.
αvβ5 Assay. ELISA plates were coated with 100 μL of
vitronectin (5 μg/mL) overnight at 4 °C in coating TS buffer.
Blocking and washing steps were performed as described for
αvβ3. Next, 3.0 μg/mL of soluble integrin αvβ5 and a serial
dilution of integrin inhibitors and the control Cilengitide were
incubated in the coated wells for 1 h at r.t. After washing three
times, the plate was incubated with 100 μL/well of primary
antibody (MAB1978) diluted 1:500 in TSB and secondary
antibody (antimouse IgG-peroxidase) at 1.0 μg/mL for 1 h at
r.t. All subsequent steps were performed as described for αvβ3.
α5β1 Assay. Flat-bottom 96-well ELISA plates were coated
overnight at 4 °C with 100 μL/well of 0.50 μg/mL of
fibronectin in carbonate buffer (see αvβ3). Blocking and
washing steps were performed as described. Next, 1.0 μg/mL
of soluble integrin α5β1 and a serial dilution of integrin
inhibitors and the control Cilengitide were incubated in the
coated wells for 1 h at r.t. After washing three times, the plate
was treated with 100 μL/well of primary antibody (CD49e) at
1.0 μg/mL (1:500 dilution) and secondary antibody (anti-
mouse IgG-peroxidase) at 2.0 μg/mL (1:385 dilution) for 1 h
at r.t. All subsequent steps were performed as described for
αvβ3.
[
99mTc(OH2)3(CO)3]+ Labeling Studies. The precursor
99mTc(OH2)3(CO)3]+ was prepared using an IsoLink Kit
[
with 1.0−2.5 GBq of Na99mTcO4 in 1−2 mL saline.36 After
neutralization to pH = 7 with HCl, quantitative labeling was
achieved by mixing an aliquot of the [99mTc(OH2)3(CO)3]+
precursor with a 0.1 mM stock solution of L1−L3 at 90 °C for
30 min and L4 at 70 °C for 25 min. Alternatively, labeling can
be performed in a microwave oven. After one minute at 130 °C,
one single product was received in yields better than 98%.
Quality control (radiochemical yield and purity determination)
of the product was determined by RP-HPLC. The identity was
assessed by comparing HPLC retention times with the
corresponding rhenium compounds (coinjection). Lipophilicity
of [99mTc(L4)(CO)3] was determined as reported previously.37
In Vivo Biodistribution Study. To establish tumor
growth, 1.5 × 107 M21 melanoma cells were injected
subcutaneously into the shoulder of female CD1 nu/nu mice
(Charles River, Sulzfeld, Germany).38 At the time of the
experiment (approximately six weeks after cell inoculation),
tumor masses ranged from 32 to 250 mg. For the
biodistribution study, 740 kBq of [99mTc(L4)(CO)3] in 100
μL of PBS were injected intravenously into the tail vein of the
M21 tumor bearing nude mice (n = 5). To demonstrate αvβ3
integrin specificity of tumor accumulation, mice (n = 3) were
coinjected with 590 μg Cilengitide per mouse (23.5 mg/kg
body weight). At 60 min post injection (p.i.), mice were
sacrificed, and the organs of interest were dissected, weighed,
and counted in a Gamma counter (Wallach, Turku). Data are
given in percent of injected dose per gram tissue [%ID/g] and
represent means standard deviation.
αIIbβ3 Assay. ELISA plates were coated overnight at 4 °C
with 100 μL/well of 10.0 μg/mL of fibrinogen in carbonate
buffer. After washing and blocking, 2.5 μg/mL of soluble
integrin αIIbβ3 and a serial dilution of integrin inhibitors and the
control molecules Cilengitide and Tirofiban were incubated in
the coated wells for 1 h at r.t. After three washing steps, wells
were treated with 100 μL/well of primary antibody (CD41b) at
29
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a
Scheme 2. Synthesis of the Orthogonally Protected BFC 2
a
(a) LiOH, 4 °C, benzaldehyde; (b) EtOH/1 M NaOH, benzyl chloroformate, −20 °C; (c) 4 M NaOH, pH = 9.5, sodium nitroprusside; (d)
PhCH2Br, Et3N; (e) MeSO2Cl, CH2Cl2, Et3N; (f) ethyl 2-(tert-butoxycarbonylamino)-2-cyanoacetate, Na[OEt], DMSO, 70 °C; (g) Boc2O,
Na[BH4], NiCl2·6H2O, MeOH; (h) Pt/C, H2, EtOH; (i) FmocCl, H2O/dioxane, Na2CO3, r.t.
Scheme 3. Structures of the Different BBN(7-14) Derivatives of L1−L3 with the L-Lys(DAP) Amino Acid Conjugated to the N-
a
Terminus (Top), Integrated into the Sequence (Middle), and Bound to the C-Terminus (Bottom)
a
Before coordinating L1−L3 to the {Re(CO)3}+ or the {99mTc(CO)3}+ moiety, respectively, the carboxylate group in the Dap chelator is protected
by an ethyl ester. Hydrolysis occurs concertedly with coordination.
30
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Scheme 4. Synthesis of RGD Peptide Analogue cyclo-(Arg-Gly-Asp-D-Tyr-Lys(DAP)) and Its Re (13) and 99mTc (13a)
Complexes: [Re(OH2)3(CO)3]+/H2O, 70°C; [99mTc(OH2)3(CO)3]+/PBS, 90 °C
Peptide Conjugation. To assess the utility of 2, three
different peptide sequences, each containing nine residues but
with a variable L-Lys(Dap) position, were selected for the
synthesis of BBN analogues. In those sequences, the single
amino acid chelate L-Lys(Dap) was introduced at the N- or C-
terminus or near the center of the peptide. The protected
amino acids were assembled sequentially on the Rink amide
resin using a standard HBTU coupling protocol. Peptides were
cleaved from the resin with simultaneous Boc protecting group
removal using a cocktail consisting of 85% TFA, 5% thioanisole,
5% phenol, and 5% water. It is important to exclude oxygen
during the cleavage reaction in order to avoid oxidation of the
methionine containing peptides. Following precipitation using
cold methyl t-butyl ether and centrifugation, the peptide−
chelator conjugates were purified by RP-HPLC (SI). Peptides
were obtained in overall yields of 45−50% based on the initial
resin loading of 0.69 mmol g−1.
The synthetic strategy for preparation of the cyclic RGD
peptide is shown in Scheme 4. Essentially, the synthesis
involved three key steps: (1) attachment to the solid support
via the α-carboxyl of Fmoc-Gly-OH, (2) linear chain formation,
and (3) head-to-tail cyclization in solution through amide bond
formation between the α-carboxyl group of Gly and the α-
amino group of Asp. Purification of the crude products with
preparative RP HPLC yielded the target compound L4 in 20%
overall yield, with >95% purity as determined by analytical
HPLC.
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of Fmoc-Lys(Dap)-OH 2
followed an orthogonal protection strategy starting from L-
lysine as shown in Scheme 2. In the first step, the ε-amino
group was masked by formation of the benzylidene imine 3.
Benzyloxycarbonyl was then introduced for protecting the α-
amino group. Hydrolytic in situ cleavage of the ε-imine gives
the N-α-benzyloxycarbonyl-L-lysine 4. In a key transformation,
the ε-amino group of 4 was converted to a hydroxyl group to
yield Cbz-protected L-α-aminoadipic acid 5. The carboxylic acid
moiety was protected to give the benzyl ester 6, and the ε-
hydroxyl function was mesylated to yield 7. Nucleophilic
displacement of the mesylate by the sodium salt of ethyl
acetamido-cyanoacetate was difficult but proceeded in reason-
able yield when carried out in DMSO.39 The resulting product
8 was catalytically reduced to Boc protected 9 in the presence
of Na[BH4]. The removal of carboxy-benzyl (Cbz) and the
benzyl (Bn) protecting groups by catalytic hydrogenation gave
the desired compound 10. The final step was Fmoc-protection
of the α-amine group under basic conditions to yield the final
product, the orthogonally protected bifunctional chelator 2.
The overall yield of the synthesis starting from L-lysine is about
10% (Scheme 2).
Although the synthesis of the orthogonally protected
bifunctional chelator 2 requires nine steps when starting from
L-Lys, the individual steps are fast and provide good yields. In
addition, reagents are cheap and the procedure is feasible on a
large scale without difficulties. Building block 2 offers numerous
possibilities for derivatization and conjugation. Since it is an α-
amino acid, it can be subjected to SPPS with essentially any
peptide sequence (vide infra). Coupling to other targeting
molecules via either the −NH2 or the −COOH group (or
both) is possible without affecting the coordinating properties
of the second function. Deprotection of the ligand yields a very
strong chelator not only for the [99mTc(CO)3]+ moiety, but
potentially also for other metals. In addition, the ligand −NH2
groups can be extended by further chelating functions, yielding
ligands with a denticity higher than three.
Rhenium Complexes. The complexes were synthesized as
references for the corresponding 99mTc-labeled peptides.
Aqueous solutions of peptides (L1−L4) were reacted with
[Re(OH2)3(CO)3]+ in H2O under N2 at 70 °C for 6−20 h to
give the Re compounds in quantitative yields (Schemes 3 and
4). The conjugates were purified by RP-HPLC and lyophilized.
ESI-MS gave the correct [m/z]+ (SI). No release of the
{ReI(CO)3} moiety from the peptides was observed,
demonstrating the stability of the complex.
99mTc-Labeling Studies. [99mTc(OH2)3(CO)3]+ was pre-
pared from Na99mTcO4 according to literature methods.36
Aqueous 99mTc solutions in the concentration range of 10−6 to
10−9 M were adjusted to pH = 7 with 1 M HCl and labeling
31
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Figure 1. HPLC traces of [M(L1)(CO)3] (14, M = Re and 14a M = 99mTc) conjugates (left) and of [M(L4)(CO)3] (13 M = Re and 13a M =
99mTc) (right).
a
Table 1. Competitive Binding Affinities of L4 and [Re(L4)(CO)3] to Different Solubilized Integrins Determined via ELISA
c
d
e
f
g
αvβ6
αvβ1
αvβ5
αIIbβ3
αvβ3
b
reference peptide
L4
30 (3.5)
254 (4.1)
540 (3.9)
6.1 (0.5)
41 (1.9)
42 (1.4)
41 (1.2)
30 (3.1)
72 (1.2)
1.5 (0.3)
440 (8.3)
432 (5.1)
0.2 (0.02)
3.4 (0.05)
7.1 (1.2)
[Re(L4)(CO)3]
a
b
Data represent IC50 values [nM] (mean of 4−6 separate experiments (SD)). Data for the respective reference peptides are cited from the
literature. Reference peptide RTDlinear IC50 = 30 nM.43 Reference peptide Cilengitide IC50 = 15 nM.44 Reference peptide Cilengitide IC50 = 50
c
d
e
nM.45 Reference peptide Tirofiban IC50 = 0.6 nM.44 Reference peptide Cilengitide IC50 = 0.5 nM35,46
f
g
efficiencies determined by reacting these stock solutions with
peptide concentration from 10−3 to 10−5 M. Over this range, L1
and L4 could be labeled with [99mTc(OH2)3(CO)3]+ at 90 °C
after 30 min in yields better than 98%. Even at a concentration
of 10−6 M, the peptides could be labeled in 85% yield after 70
min, underlining the efficacy of the Dap chelator. In the
presence of 0.1 M cysteine or histidine at 37 °C, no trans-
metalation was found after 24 h, further confirming the
chemical robustness of the Dap complex.27 It should be
emphasized at this point that the labeling yield did not depend
on the absolute amount of 99mTc activity, an observation in
agreement with the pseudo first-order labeling kinetics in Dap.
Typical specific activities as achieved in our labeling studies
were on the order of 1.1 TBq/μmol. Dap is, thus, suitable for
labeling biomolecules to very high specific activities. It is
important to note that specific activities are calculated from the
crude radiolabeling solution and that, due to the very low
amounts of Dap-conjugate needed for efficient [99mTc-
(OH2)3(CO)3]+ complexation, an HPLC separation of 99mTc-
labeled product from unreacted precursor is not necessary. The
identity of the 99mTc-labeled conjugates was confirmed by
HPLC coinjection with the corresponding rhenium complexes
(Figure 1). Due to the close structural similarity between Re
and Tc homologues, retention times should be close to
identical.
In Vitro Evaluation. For an exemplary evaluation of the
effect of Lys-by-Lys(Dap) substitution on receptor binding
affinity of cyclic RGD analogues, we investigated the binding
affinity of [Re(L4)(CO)3] to different clinically relevant
integrin receptor subtypes.40,41
The integrins with the highest documented relevance for
imaging applications contain the αv subunit, especially the αvβ3
and αvβ5 subtype. The former is known to be overexpressed on
many tumor types and tumor neovasculature, but is also
expressed at lower levels in noncancerous tissues.42 Table 1
summarizes the binding affinities (IC50 in nM) of [Re(L4)-
(CO)3] as well as of the uncomplexed labeling precursor L4 to
these and three other integrin receptor subtypes.
The rhenium reference peptide [Re(L4)(CO)3] and the
uncomplexed labeling precursor L4 display similar IC50 values
for all integrin subtypes investigated. However, in the case of
the αvβ6, αvβ5, and αvβ3 integrins, complexation with the
[Re(CO)3]+ fragment leads to a loss in binding affinity by a
factor of 2. Nevertheless, binding affinity of L4 integrates well
into a series of different RGD-based precursors for 99mTc-
labeling such as Pz1-RGD, HYNIC-RGD, Cys-RGD, and L2-
cRGD with IC50 values of 3, 6, 6.6, and 11.8 nM, respectively,
in a comparable assay.47 The αvβ3-integrin affinity of [Re(L4)-
(CO)3] is comparable to that of other clinically used
radiolabeled RGD-analogues such as [18F]Galacto-RGD. [18F]-
Galacto-RGD shows an affinity of 5 nM to the immobilized
αvβ3 receptor, but significantly higher αvβ3 selectivity (IC50
(αvβ5) = 1000 nM, IC50 (αIIbβ3) = 6000 nM) than the peptides
investigated in this study.48
The results obtained in the binding study using M21 cells
and [125I]echistatin as the radioligand show the same tendency:
Re(CO)3-complexation of L4 leads to a loss in binding affinity
(IC50 (L4) = 242 nM, IC50 ([Re(L4)(CO)3]) = 866 nM),
albeit somewhat more pronounced than observed when using
immobilized integrin. The affinity determined for [18F]Galacto-
RGD in the same assay is 319 nM.49
In Vivo Biodistribution Study. The biodistribution data
obtained for [99mTc(L4)(CO)3] in M21 melanoma bearing
nude mice are summarized in Table 2.
[
99mTc(L4)(CO)3] shows rapid clearance from the circu-
lation and no particular predominance of renal vs hepatobiliary
clearance or vice versa, but modest accumulation in all
excretion organs. This represents a major advantage of this
new Lys(Dap)-coupled RGD analogue over previous deriva-
tives using other BFCs for [99mTc(CO)3]+ complexation.50 For
example, [99mTc(CO)3]Pz1-cRGD, in which pyrazolyl function-
alities serves for [99mTc(CO)3]+ complexation, shows 5-fold
higher hepatic and intestinal accumulation. This may be due to
the significantly reduced lipophilicity of [99mTc(L4)(CO)3] as
compared to [99mTc(CO)3]Pz1-cRGD (log P = −1.82 vs
−0.92). Surprisingly, the accumulation of [99mTc(L4)(CO)3] in
the excretion organs is as low as or even lower than that of
32
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Table 2. Biodistribution of [99mTc(L4)(CO)3] in M21
Melanoma Bearing Nude Mice 60 min p.i.
tripod ligand is one of the smallest and most efficient tridentate
ligands for [99mTc(CO)3]+ complexation described so far, and is
suited for labeling not only peptides, but also other
biomolecules or pharmacophores. As HYNIC, it has the
potential to become a standard in 99mTc-based molecular
imaging, but unlike for HYNIC-based 99mTc complexes, the
authenticity of the conjugates is well-defined.
a
organ
blood
control
blocking study
tumor/organ ratio
0.37 0.02
0.34 0.02
0.78 0.13
0.63 0.13
0.24 0.01
2.10 0.50
1.02 0.15
1.89 0.45
2.02 0.29
0.18 0.01
1.41 0.16
0.31 0.25
0.20 0.04
0.41 0.06
0.22 0.04
0.10 0.01
1.42 0.27
0.35 0.23
1.31 0.77
1.22 0.22
0.07 0.02
0.25 0.14
3.8 0.5
4.2 0.5
1.8 0.4
2.3 0.5
5.9 0.7
0.7 0.2
1.4 0.3
0.8 0.2
0.7 0.1
7.7 1.0
heart
lung
spleen
pancreas
liver
ASSOCIATED CONTENT
■
S
* Supporting Information
stomach
intestines
kidney
muscle
tumor
Additional analytical data such as 13C NMR, HPLC traces, and
mass spectra and general synthetic procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
a
AUTHOR INFORMATION
Control animals (n = 5) were injected with tracer only, for the
■
blocking study (n = 3), the tracer and 590 μg Cilengitide per mouse
(23.5 mg/kg body weight) were coinjected. Data are given as %ID/g
and are means SD.
Corresponding Author
*E-mail: ariel@aci.uzh.ch.
Notes
The authors declare no competing financial interest.
[
99mTc]EDDA/HYNIC-cRGD, which shows an even lower log
P of −3.57.47 This emphasizes once more the observation that,
especially in the case of 99mTc-labeled peptide radiopharma-
ceuticals, the labeling method itself rather than the physical
parameter “hydrophilicity” governs excretion pathways from
the circulation.
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■
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CONCLUSION
■
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