Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine for peptide conjugation and 64Cu radiolabelling

Article abstract of DOI:10.1039/b902210d

Copper-64 shows promise as both a suitable PET imaging and therapeutic radionuclide due to its nuclear characteristics. Stable attachment of radioactive ⁶⁴Cu²⁺ to targeted imaging probes requires the use of a bifunctional chelator. S

Full text of DOI:10.1039/b902210d

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Synthesis of a novel bifunctional chelator AmBaSar based on sarcophagine
for peptide conjugation and 64Cu radiolabelling†
Hancheng Cai, John Fissekis and Peter S. Conti*
Received 3rd February 2009, Accepted 17th April 2009
First published as an Advance Article on the web 22nd May 2009
DOI: 10.1039/b902210d
Copper-64 shows promise as both a suitable PET imaging and therapeutic radionuclide due to its
nuclear characteristics. Stable attachment of radioactive 64Cu2+ to targeted imaging probes requires the
use of a bifunctional chelator. Sarcophagine (Sar) ligands coordinate the metal ion 64Cu2+ within the
multiple macrocyclic rings comprising the cage structure, yielding extraordinarily stable complexes that
are inert to dissociation of the metal ion in vivo. Several 64Cu labelled RGD derivatives have been
applied in imaging of the anb3 integrin receptor expression during tumour angiogenesis. In order to
design and develop novel molecular imaging probes containing RGD and Sar ligands, we designed a
novel versatile Sar cage-like bifunctional chelator named AmBaSar, synthesized using a conventional
synthetic strategy. Conjugation with the cyclic peptide RGD, and subsequent labelling with 64Cu,
provided a new PET probe 64Cu-AmBaSar-RGD for imaging the anb3 integrin receptor.
and bioactive molecule to form the 64Cu-radiopharmaceutical.
Optimization of the binding between the copper-64 and the BFC
is crucial for achieving stable and high uptake of targeted 64Cu-
radiopharmaceuticals.5
Introduction
Molecular imaging is an emerging technology that allows for
visualization of interactions between molecular probes and bio-
logical targets. Various modalities, which include nuclear medicine
imaging, such as positron emission tomography (PET) and sin-
gle photon emission tomography (SPECT), functional magnetic
resonance imaging (fMRI), magnetic resonance spectroscopy
(MRS), optical imaging including bioluminescence and fluores-
cence, targeted ultrasound, and the others have been applied.1–3
The advancement of molecular imaging is driven mainly by the
development of smart imaging probes targeted to specific receptors
and enzymes.1,4
The stability of 64Cu–radiopharmaceuticals in vivo is a critical
factor for optimal ligand design. A significant effort has been
devoted to the development of ligands that can stably chelate
64Cu2+ in vivo. Two of the most common chelators studied
have been the macrocyclic ligands DOTA (1,4,7,10-tetraazacyclo-
dodecane-N,N¢,N¢¢,N¢¢¢-tetraacetic acid) and TETA (1,4,8,11-
tetraazacyclotetradecane-N,N¢,N¢¢,N¢¢¢-tetraacetic acid). Recent
studies have shown that these commonly used BFCs are un-
stable in vivo due to increased dissociation relative to more
recently developing BFCs, such as the cross-bridged tetraamine
ligands, the sarcophagine ligands and the triaminocyclohexane
ligands.6–9 Among these new BFCs, the Sar ligands containing
hexaazamacrobicyclic cages have been developed by Sargeson
and co-workers, and have recently been used for the preparation
of 64Cu–radiopharmaceuticals by Smith et al.10–13 These ligands
coordinate the metal ion Cu2+ within the multiple macrocyclic
rings comprising the Sar cage structure, yielding extraordinarily
stable complexes that are inert to dissociation of the metal ion.
A cage-like hexaazamacrobicyclic sarcophagine (Sar, Fig. 1A),
(NH2)2-Sar (Diamsar, Fig. 1B), and their derivatives, have shown
the potential for biology applications, including developing
imaging and therapeutic agents.14 Recently, a derivative of the
DiamSar ligand, named SarAr (Fig. 1C), has been conjugated to
antibodies and radiolabelled with 64Cu. The resulting 64Cu-SarAr
immunoconjugates have shown high specific activity, antigen
binding affinity, and in vivo target specificity to neuroblastoma
and melanoma, with minimal uptake in normal tissues.12,13 The
cage-like BFC Sar ligands are unique in their ability to selectively
label 64Cu2+ rapidly over a wider range of pH values.11 The cage-
like Sar ligand is not only flexible for the incorporation of a
range of functional linker groups and quantitatively complexing
64Cu2+ at low concentrations, but also is adaptable for kit
PET, micro-PET and PET/CT, are state-of-the-art nuclear
medicine imaging modalities, which use nano- to picomolar
concentrations of the corresponding probes (radiotracers) to
achieve images of biological processes within the living system.
The selection of the proper radionuclide and synthetic approach
for radiotracer design are critical.1 Positron-emitting isotopes
frequently used include 11C and 18F. Recently, non-traditional
PET radionuclides have gained considerable interest because of
increased production and availability, such as 124I, 68Ga, 86Y and
62/64Cu. One of them, 64Cu, shows promise as both a suitable
PET imaging and therapeutic radionuclide due to its nuclear
characteristics (t1/2 = 12.7 h, b : 17.4%, Eb+max = 656 keV; b-: 39%,
+
Eb-max = 573 keV), and the availability of its large-scale production
with high specific activity.3 The stable attachment of radioactive
64Cu2+ to targeted imaging probes requires the use of a bifunc-
tional chelator (BFC), which is used to connect a radionuclide
Molecular Imaging Center, Department of Radiology, Keck School of
Medicine, University of Southern California, Los Angeles, CA 90033, USA.
E-mail: pconti@usc.edu; Fax: +1 323-442-5778; Tel: +1 323-442-5940
† Electronic supplementary information (ESI) available: 1H-NMR spectra
of Cu-DiamSar, Cu-AmBaSar, AmMBSar, and AmBaSar; MS result of
AmBaSar-RGD; HPLC results of AmBaSar, AmBaSar-RGD and 64Cu-
AmBaSar-RGD. See DOI: 10.1039/b902210d
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“M-(X,Ysar)”, where M represents the metal ion, and X and Y
are the substituents in the 1- and 8-positions of the Sar cage.
(1,8-Dinitro-Sar)cobalt(III) trichloride = [Co(DiNosar)]Cl3; (1,8-
diamine-Sar)cobalt(III) pentachloride = [Co(DiAmSar)]Cl5; (1,8-
diamine-Sar)copper(II) tetrachloride = [Cu(DiAmSar)]Cl4; (1-
amine,8-(aminomethyl)-4¢-methylbenzoate-Sar)copper(II) = [Cu-
(AmMBSar)]2+; (1-amine,8-(aminomethyl)-benzoic acid-Sar)-
copper(II) = [Cu(AmBaSar)]2+; (1,8-(aminomethyl)-4¢-methyl-
benzoate-Sar)copper(II) = [Cu(DiMBSar)]2+.
Fig. 1 Chemical structures of Sar, DiamSar, SarAr and AmBaSar.
Experimental
formulations. However, there are only a few reports that describe
the complexation, stability and biodistribution of the 64Cu Sar
complexes. Unfortunately, only one compound, a SarAr BFC for
64Cu2+ conjugated with a monoclonal antibody (mAb), has been
applied in PET imaging.13
Methods and materials
1H NMR spectra were obtained using a Varian Mercury 400 MHz
instrument (USC NMR Instrumentation Facility), and the chemi-
cal shifts were reported in ppm on the d scale relative to an internal
TMS standard. Microanalyses for carbon, hydrogen, nitrogen
and chlorine, cobalt, copper were carried out by the Columbia
Analytical Services, Inc (Tucson, AZ). Mass spectra using LC-MS
were operated by the Proteomics Core Facility of the USC School
of Pharmacy. Thin-layer chromatography (TLC) was performed
on silica gel 60 F-254 plates (Sigma-Aldrich) using a mixture
solution of 70% MeOH and 30% aqueous NH4OAc (NH4OAc
solution is 20% by weight) as the mobile phase. Ion-exchange
chromatography was performed under gravity flow using Dowex
50WX2 (H+ form, 200–400 mesh) or SP Sephadex C25 (Na+
form, 200–400 mesh) cation exchange resins. All evaporations were
performed at reduced pressure (ca. 20 Torr) using a Bu¨chi rotary
evaporator.
HPLC was accomplished on two Waters 515 HPLC pumps, a
Waters 2487 absorbance UV detector and a Ludlum Model 2200
radioactivity detector, operated by Waters Empower 2 software.
Purification of the conjugate AmBaSar-RGD was performed on
a Phenomenex Luna C18 reversed phase column (5 mm, 250 ¥
10 mm); the flow was 3.2 mL min-1, with the mobile phase solvent
A (12% acetonitrile in water), and the absorbance monitored
at 254 nm. The analytical HPLC was done on a Phenomenex
Luna C18 reversed phase column (5 mm, 250 ¥ 4.6 mm) and
monitored using a radiodetector and UV at 218 nm. The flow was
1.0 mL min-1, with the mobile phase starting from 99% solvent B
(0.1% TFA in water) and 1% solvent C (0.1% TFA in acetonitrile)
(0–1 min) to 70% solvent B (0.1% TFA in water) and 30% solvent
C (0.1% TFA in acetonitrile) (1–30 min).
The development of other linkage strategies for Sar is of
interest to our laboratory. Primary amines and carboxylic acids
are the most common examples of pendent arms that have been
introduced to the DiamSar periphery. Direct introduction the
carboxymethyl entities to DiamSar may be complicated and bring
many by-products due to the side reactions on the secondary
amines.15 Likewise, using benzoic acid to functionalize DiamSar
is an option. Based on this, we have designed a new Sar cage-
like BFC named AmBaSar (Fig. 1D) by introducing benzoic
acid to DiamSar. Tagged small peptides have attracted much
attention recently as molecular imaging probes due to their
relatively low immunogenicity, good pharmacokinetic properties,
and binding affinities.16 For example, conjugates containing the
cyclic peptide Arg-Gly-Asp (RGD), bind to cancer cells and/or
neoplastic vascular endothelial cells via the anb3 integrin receptor,
andtherefore have potentialas angiogenesis-related diagnostic and
therapeutic agents.17,18 Several 64Cu labelled RGD derivatives have
been applied in imaging of the anb3 integrin receptor expression
during tumour angiogenesis.17–19 However, work remains to be
done to optimize the pharmacokinetics and dynamics of these
agents for use in animal and human studies. A reasonable next
step is to select a new sarcophagine chelator for 64Cu labelling
RGD. In this study, we have designed a novel molecular imaging
probe containing bioactive cyclic peptide RGD with a versatile Sar
cage-like BFC AmBaSar. We have synthesized AmBaSar using a
conventional synthetic strategy, and applied it conjugating the
small cyclic peptide RGD for 64Cu radiolabelling.
All reagents and solvents were purchased from Sigma-Aldrich
Chemicals (St. Louis, MO, USA) and used without further
purification unless otherwise stated. N-hydroxysulfosuccinimide
sodium salts (SNHS) and N-ethyl-N¢-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl) were obtained from the
Sigma-Aldrich Chemical Co. The cyclic RGDyK peptide was
purchased from Peptides International, Inc (Louisville, KY, USA).
64CuCl2 was purchased from MDS Nordion (Vancouver, BC,
Canada). Water was purified using a Milli-Q ultra-pure water
system from Millipore Corp. (Milford, MA, USA).
Abbreviations
The IUPAC names for the cage ligands are long and complicated.
In this study, the names have been abbreviated as follows:
3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane = “sarcophagine”
or “Sar”; 1, 8-diamine-3,6,10,13,16,19-hexaazabicyclo[6.6.6]ico-
sane = “DiamSar”; 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaa-
zabicyclo[6.6.6]icosane-1,8-diamine = “SarAr”; methyl-4-((8-am-
ino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1-ylamino)met-
hyl)benzoate = “AmMBSar”; 4-((8-amino-3,6,10,13,16,19-hexaa-
zabicyclo[6.6.6]icosane-1-ylamino)methyl)benzoic acid = “AmBa-
Sar”.
The bifunctional chelator AmBaSar synthesis and characterization
For complexes of Sar ligands, we have used nomencla-
ture which represents these M-hexaamine cage complexes by
Synthesis of [Co(DiNoSar)]Cl3 (1). To
tris(ethylenediamine)cobalt(III) chloride dehydrate (1.2 g,
a solution of
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3.0 mmol) in water (4.0 mL) was added nitromethane (0.7 g,
12 mmol) and aqueous formaldehyde ◦(37%, 2.4 g, 30 mmol).
The resulting solution was cooled to 4 C on an ice–water bath.
yielded a blue precipitate. The blue solid was cooled, filtered,
and washed with ethanol (3 ¥ 5 mL), and dried, yielding a blue
1
crystalline product 4 (0.53 g, 97%). H-NMR (D2O): d 3.20 (s,
◦
Aqueous NaOH (4.0 M, 3.0 mL) was cooled to 4 C and mixed
12 H, NCH2CH2N); 2.90 (s, 12 H, NCCH2N). MS calcd for
+
with the resulting solution above. The combined solution was
stirred on ice-water bath where it rapidly turned deep violet-brown
from the initially orange colour, and the reaction temperature was
raised to room temperature 23–25 ◦C. After 90 min, the reaction
was quenched by the addition of HCl (6.0 M, 2.0 mL). The orange
precipitate formed was collected by filtration after cooling on ice
for 2 h, washed with methanol, and dried to provide 1 (1.46 g,
C14H33CuN8 [M + 1 - 4HCl - 5H2O] m/z 376.2, found 376.0.
Elemental analysis calculated for C14H46Cl4CuN8O5, requires C
27.48, H 7.58, N 18.31, Cl 23.17, Cu 10.38. Found: C 27.72, H
7.17, N 17.87, Cl 22.40, Cu 9.71.
Synthesis of [Cu(AmMBSar)](CH3COO)2·5H2O (5). [Cu(Di-
AmSar)]Cl4·5H2O (0.73 g, 1.2 mmol) was dissolved in dry ethanol
(30 mL), followed by addition of methyl 4-formylbenzoate (0.28 g,
1
90.2%). H-NMR (D2O): d 3.55–3.65 (d, 6 H, NCCH2N); 3.15–
˚
1.7 mmol), dried/activated 4 A molecular sieves (1.0 g) and glacial
3.35 (d, 6 H, NCCH2N); 3.00–3.10 (d, 6 H, NCH2CH2N); 2.55–
acetic acid (60 mL). The resulting solution was stirred for 3 h under
argon gas, followed by addition of sodium cyanoborohydride
(0.82 g, 14 mmol). The reaction mixture continued to stir under
argon gas for 4 d at room temperature 20–25 ◦C. The mixture
was filtered, and filtrate was evaporated to dryness and extracted
with ethyl acetate (3 ¥ 15 mL), dried and then diluted to 300 mL.
It was placed onto a SP Sephadex C25 column and eluted with
sodium citrate (0.1 M, 400 mL) and a wide blue band formed.
Increasing the sodium citrate concentration (0.3 M, 500 mL)
resulted in three blue bands eluting in order as [Cu(DiamSar)]2+,
[Cu(AmMBSar)]2+ (compound 5), and [Cu(DiMBSar)]2+ by TLC
monitoring. The second band (compound 5 solution, 100 mL) was
isolated and diluted with water (10 fold, 1.0 L), and placed onto
another SP Sephadex C25 column. A single blue band eluted with
sodium acetate (1.0 M, 200 mL), was evaporated to dryness and the
residue extracted with 2-propanol (3 ¥ 50 mL). Fine white crystals
of sodium acetate were separated, filtered and the process of
evaporation and extraction repeated 3 times. The final residue was
dried in vacuo to a dark blue solid 5 [Cu(AmMBSar)](Ac)2·5H2O
(246.5 mg, 27.9%). 1H-NMR (D2O): d 8.0–7.9 (d, 2 H, aromatic);
7.48–7.42 (d, 2 H, aromatic); 4.70 (s, 3 H, OCH3); 4.02 (s, 2 H,
CH2-Ar); 3.54–3.30 (m, 12 H, NCH2CH2N); 2.93–2.78 (m, 12 H,
NCCH2N). MS calcd for C23H43CuN8O2 [M + 1 - 2HAc - 5H2O]
2.65 (d, 6 H, NCH2CH2N). MS: calcd for C14H31CoN8O4 [M + 1 -
3HCl] m/z 431.2, found 431.5. Elemental analysis calculated
for C14H30Cl3CoN8O4, requires C 31.15, H 5.60, N 20.76, Cl
19.71, Co 10.92. Found: C 30.90, H 5.46, N 20.13, Cl 20.80, Co
10.70.
+
Synthesis of [Co(DiamSar)]Cl5·H2O (2). [Co(DiNoSar)]Cl3
(2.0 g, 3.7 mmol) was dissolved in deoxygenated water (100 mL)
under N2 atmosphere. Zinc dust (2.3 g, 35 mmol) was added into
the solution with stirring for 5 min, followed by addition of HCl
(6 M, 15 mL) dropwise. The resulting solution continued to stir
for an additional 60 min under a N2 atmosphere. The N2 flow
was stopped and 30% H2O2 (1.0 mL) was added. The resulting
solution was warmed for 15 min on 75 ◦C water bath, then cooled
and placed on a Dowex 50WX2 cation exchange column and
washed with water (120 mL), followed by HCl (1.0 M, 120 mL).
The complex was then eluted with HCl (3.0 M, 400 mL) and
the yellow elute was collected and dried under vacuum to yield 2
(1.78 g. 87%). 1H-NMR (D2O): d 3.30–3.10 (m, 12 H, NCCH2N);
2.50–2.65 (m, 12 H, NCH2CH2N). MS calcd for C14H35CoN8 [M +
+
1 - 5HCl - H2O] m/z 371.2, found 371.7. Elemental analysis
calculated for C14H38Cl5CoN8O, requires C 29.46, H 6.71, N 19.63,
Cl 31.06, Co 10.33. Found: C 29.06, H 6.73, N 19.04, Cl 25.30, Co
9.50.
+
m/z 526.3, found 526.8.
Synthesis of DiamSar·5H2O (3). Co(DiamSar)]Cl5·H2O
(3.58 g, 6.3 mmol), NaOH (0.58 g, 14.5 mmol, sufficient to neutral-
ize the protonated primary amino groups) and cobalt(II) chloride
hexahydrate (1.6 g, 6.4 mmol) were dissolved in deoxygenated
water (50 mL) under nitrogen. Sodium cyanide (5.60 g, 114 mmol)
was added to the resulting solution. The reaction mixture was
heated to 70 ◦C being stirred under nitrogen until the solution
had become almost colourless (overnight). This final solution was
taken to dryness under vacuum, with the residue extracted with
boiling acetonitrile (3 ¥ 25 mL). The total extract was filtered,
reduced under vacuum to a white solid, and cooled to -10 ◦C to
precipitate white crystals of th◦e product. Drying in vacuo provided
3 (1.72 g, 58.5%). Mp 91–94.0 C. 1H-NMR (D2O): d 2.51 (s, 12 H,
NCCH2N); 2.42 (s, 12 H, NCH2CH2N). MS calcd for C14H35N8
Synthesis of AmMBSar (6). Sodium borohydride (150 mg) was
dissolved in 0.4 mL water and stirred under a nitrogen atmosphere,
following addition of Pd/C (60 mg) in 1.0 mL water. Compound
5 (164 mg) was dissolved in sodium hydroxide (3 mL; 1% NaOH)
and added dropwise to the above mixture solution. Stirring was
◦
continued under nitrogen at 25 C until the colour turned from
blue to clear. The resulting solution was filtered (0.22 mm) and
the filtrate was collected in an ice-cooled glass vial. Concentrated
hydrochloric acid was added dropwise (50 mL) to the cooled
solution until gas evolution ceased (~ 450 mL, HCl). The solution
was acidified to pH 4–6, and then dried under vacuum to provide
1
AmMBSar, 6 (56 mg, 47.6%). H-NMR (D2O): d 7.97–7.92 (d,
2 H, aromatic); 7.50–7.45 (d, 2 H, aromatic); 4.65 (s, 3 H, OCH3);
4.08 (s, 2 H, CH2-Ar); 3.45–3.20 (m, 12 H, NCH2CH2N); 3.12–
2.98 (m, 12 H, NCCH2N); 1.98–1.85 (m, 9 H, NH). MS calcd for
C23H42N8O2 [M + 1] + m/z 463.0, found 462.3.
+
[M + 1 - 5H2O] m/z 315.3, found 315.8. Elemental analysis
calculated for C14H44N8O5, requires C 41.56, H 10.96, N 27.70.
Found: C 41.53, H 10.28, N 27.12.
Synthesis of AmBaSar (7). Compound 6 (46.2 mg, 0.1 mmol)
was dissolved in methanol (3 mL) and water (1.0 mL), followed
by addition of NaOH (1.5 mL, 0.1 M). The resulting solution was
refluxed and stirred for 5 h, then neutralized with HCl (1.0 M)
to pH 6–7. Drying the solution under reduced pressure formed
Synthesis of [Cu(DiamSar)]Cl4·5H2O (4). CuCl2·2H2O (0.17 g,
1.0 mmol), was dissolved in 10 mL water, followed by adding
3 (0.41 g, 1.0 mmol). The solution was acidified to pH = 4.0
with HCl (0.1 M), and stirred overnight. Evaporation on heating
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solids, which were dissolved with hot MeOH (3 ¥ 2 mL). The total
extract was filtered and dried under vacuum to yield AmBaSar, 7
(36 mg, 80.2%). 1H-NMR (D2O): d 7.76–7.67 (m, 2 H, aromatic);
7.45–7.37 (m, 2 H, aromatic; 3.66 (s, 2 H, NCH2C) 3.26–3.04 (m,
12 H, NCCH2N); 2.99–2.84 (m, 12 H, NCH2CH2N); 1.84–1.77
aromatic carboxyl, which can conjugate a cyclic peptide containing
the RGD motif in addition to a lysine for conjugating to the
chelator, to form BFC AmBaSar with minimum modification to
SarAr. The novel BFC AmBaSar can efficiently label 64Cu2+ due to
the provision of a three-dimensional hexa-aza cage which increases
thermodynamic and kinetic stability to complex 64Cu2+ or other
metal ions, while allowing the aromatic linker with carboxyl acid
group to conjugate with the amine of lysine in the cyclic peptide
containing the RGD motif. The AmBaSar-RGD can possibly be
used to form new nuclear, MRI, and optical probes by complexion
with other appropriate metal radioisotopes or paramagnetic metal
ions using similar preparations. We anticipate that the AmBaSar
can be applied to the other peptides or biomolecules, since the
chemistry for conjugating AmBaSar to other peptides is similar to
that described for the cyclic peptide RGD.
+
(m, 9 H, NH). MS calcd for C23H41N8O2 [M + 1] m/z 449.3,
found 449.7.
AmBaSar conjugating peptide RGD and radiolabelling
Synthesis of AmBaSar-RGD (8). AmBaSar was activated by
EDC at pH 5.5 for 30 min (4 ◦C), with a molar ratio of
AmBaSar : EDC : SNHS = 5 : 5 : 4. Typically, 15.0 mg of AmBaSar
(30 mmol) was dissolved in 500 mL of water. Separately, 5.76 mg of
EDC (30 mmol) was dissolved in 500 mL of water. The two solutions
were mixed, and 0.1 M NaOH (250 mL) was added to adjust the
pH to 4.5. SNHS (5.2 mg, 24 mmol) was then added to the stirring
mixture on an ice-bath, followed by 0.1 M NaOH (50 mL) to
finalize the pH to 5.4. The reaction was allowed to stir for 30 min
An efficient synthesis of the bifunctional chelator AmBaSar
was shown in Scheme 1. It is worth noting that the degree of
hydration of the synthesized compounds in this study is dependent
on the method of purification. Although compounds 1–4 can be
prepared following literature methods using the cobalt complexes
as starting materials,20,21 the reported syntheses of compound
1–4 are incomplete, with compounds not fully characterized.
Here, the comprehensive and detailed synthetic procedures with
full characterization of these compounds (including MS, element
analysis, 1H-NMR) are reported. Tris(ethylenediamine)cobalt(III)
chloride initiated the encapsulation process using the reactive
nucleophile nitromethane and formaldehyde in alkaline solution
at room temperature to form a hexa-aza cage containing Co(III)
compound 1, which was then reduced with zinc dust in acid
solution to give the very stable Co(III) DiamSar complex 2. The
Co(III) ion of compound 2 can be removed by reduction with
high concentrations of hydrochloric or hydrobromic acid at high
temperatures (130–150 ◦C) or excess cyanide ion to yield the
free cage. Here, we used excess cyanide ion to remove the metal
ion in compound 2 to form compound 3 in good yield (58.5%).
The six nitrogen donor atoms of the hexa-aza cage allows strong
binding to many metal ions, such as Cu2+, so compound 3 was
easy to complex with metal ion Cu2+, yielding compound 4 under
weak acid conditions. The compounds 1–4 were characterized by
◦
at 4 C. The theoretical concentration of active ester AmBaSar-
OSSu was calculated to be 24 mmol. Cyclic RGD peptide (2.5 mg,
4.0 mmol) dissolved in 500 mL (5.0 mg mL-1) of water was cooled
to 4 ◦C and added to the AmBaSar-OSSu reaction mixture.
The pH was adjusted to 8.6 with 0.1 M NaOH (280 mL). The
reaction was allowed to proceed overnight at room temperature
(20–25 ◦C). The AmBaSar-RGD conjugate was purified by semi-
preparative HPLC. The peak containing the RGD conjugate was
collected, lyophilized, and dissolved in water at a concentration of
1.0 mg mL-1 for use in radiolabelling reactions.
Synthesis of reference compound [Cu-AmBaSar-RGD](Ac)2·
2HAc. The AmBaSar-RGD (1.0 mg) was dissolved in 0.5 mL of
a 0.1 M ammonium acetate–0.80 mM copper(II) acetate solution.
The mixture was stirred at 37 ◦C for 40 min and allowed to cool
to room temperature. The crude Cu-AmBaSar-RGD solution was
purified and quantified by HPLC. MS calcd for C58H94N16O17Cu
[M + 1] + m/z 1352.0, found 1351.9.
Radiolabelling of 64Cu-AmBaSar-RGD. [64Cu]acetate (64Cu-
(OAc)2) was prepared by adding 111 MBq (3 mCi) of 64CuCl2
in 0.1 M HCl into an 1.5 mL microfuge tube containing 300 mL
0.1 M ammonium acetate (pH 5.0), followed by mixing using a
mini vortex and incubating for 15 min at room temperature. The
AmBaSar-RGD (1–2 mg in 100 mL 0.1 M ammonium acetate) was
labelled with 64Cu(OAc)2 by addition of 1–3 mCi of 64Cu. The
chelation reaction was performed in 0.1 M sodium acetate buffer,
pH 5.0, for 60 min at room temperature (23–25 ◦C). Labelling
efficiency was determined by HPLC. 64Cu-AmBaSar-RGD was
purified by radio-HPLC. The eluant was evaporated and the
activity reconstituted in saline, followed by passage through a
0.22 mm Millipore filter into a sterile dose vial for use in animal
experiments.
1
elemental analysis, MS and H-NMR, the results are consistent
with those reported previously.20,21 The yields obtained using the
modified procedures shown here were similar to or better than
those previously reported.
It is challenging to functionalize the apical primary amines of
DiamSar 3 directly because there are 2 primary and 6 secondary
amines in DiamSar, which potentially could create many by-
products and difficulty during purification. The initial formation
of copper(II) complex of DiamSar 4 was an attractive solution as
it would serve to tie up the 6 secondary amines of DiamSar, and
also permit the tracking of the resultant Cu(II) complexes on the
ion exchange columns.10 Structural studies have confirmed that
it is possible to exploit the relatively low nucleophilicity of the
Cu(II) complex of DiamSar in acylation and alkylation reactions
leading to a variety of functionalized cage amine complexes.22,23
We utilized the hydride reducing agent sodium cyanoborohydride
(NaBH3CN) for reduction due to its stability in relatively strong
acid solutions (about pH 3), its solubility in hydroxylic solvents
such as ethanol, and its different selectivities at different pH values.
At pH 3–4 it reduces the imine to an amine efficiently, while this
Results and discussion
Design, synthesis, and characterization of bifunctional chelator
AmBaSar
In order to design a BFC such as SarAr for 64Cu labelling and
conjugating with RGD, it was necessary to modify the linkage
of DiamSar. We replaced the aromatic amine of SarAr with an
5398 | Dalton Trans., 2009, 5395–5400
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Scheme 1 Synthesis of bifunctional chelator AmBaSar.
reduction becomes very slow at higher pH values.24 Hence, the
reducing reaction of the cage amine of compound 4 with aromatic
methyl-4-formylbenzoate under NaBH3CN ethanol acid solution
can yield aromatic functionalization of the cage amine compound
5 and the by-product bis-4-formylbenzoate diamsar complex. This
reaction took a significant amount of time (4 days) and yield
was 27.9% due to low nucleophilicity. Separation was carried out
by ion exchange chromatography. Compound 6 was achieved by
demetallation of compound 5, followed by alkaline hydrolysis
to produce the target compound 7 AmBaSar according to the
reported method.25
Initially, we planned to use Fmoc protection of the free primary
amine of AmBaSar in order to apply a solid-phase peptide
synthesis (SPPS), in the event that the primary amine might
perturb the conjugation with peptides. But this reaction did not
happen as we expected or the yield was too low to be detected (data
not shown). This likely means the primary amine of AmBaSar is
inactive under these conditions. The reason for the poor reactivity
of the primary amine in AmBaSar may be due to the structure of
the cage hindering the reaction between Fmoc-Cl with the primary
amine. This result is consistent with the inability of DiamSar
to directly efficiently conjugated antibodies or proteins.12,26 This
provides additional proof that DiamSar should be functionalized
and the suitable linker group should be sufficient length (~6 atom
lengths) and rigidity incorporating a reactive group that could be
readily attached to a range of target agents, but not interfere with
DiamSar’s efficient and selective complexation of metal ion like
Cu2+, as well as the target agent’s biological activity.10 AmBaSar
appears to meet these requirements.
AmBaSar conjugating cyclic peptide RGD and 64Cu radiolabelling
The bifunctional chelator AmBaSar contains one carboxyl
group and an inactive primary amine, which means that it is a
mono-functional molecule that can react with a cyclic peptide
containing the RGD motif in addition to a lysine for conjugating
to the chelator. Here, we used a simple procedure for conjugation
the cyclic RGD and BFC AmBaSar and common procedures for
64Cu radiolabelling conjugates.27–29 Scheme 2 depicts AmBaSar
conjugating cyclic RGD and 64Cu radiolabelling. The cyclic RGD
conjugate AmBaSar-RGD was synthesized in 80% yield and
purified by semi-preparative HPLC. Analytical HPLC found
the retention time of AmBaSar-RGD to be 3.8 min, whereas
cyclic RGD peptide eluted at 25 min under the same condition.
AmBaSar-RGD was analyzed by mass spectrometry, found m/z =
1049.3 for [M + H] + (M = C50H80N16O9) and 1089.7 for [M + K]+.
The peptide–chelator conjugate AmBaSar-RGD can be com-
plexed with 64Cu to form a new PET tracer for imaging the anb3
integrin receptor. AmBaSar–RGD was labelled with 64Cu in 0.1 M
ammonium acetate (pH 5.0) solution at room temperature (25 ◦C)
for 1 h. The free 64Cu-acetate was eluted at 3.2 min, while 64Cu-
AmBaSar-RGD was eluted at 15.8 min by analytical HPLC, which
was confirmed by cold Cu-AmBaSar-RGD. The radiochemical
yield obtained was ≥80% and the radiochemical purity was ≥95%.
We did not optimize the radiolabelling here because our overall
objective was to demonstrate the feasibility of preparing our novel
BFC. We are currently conducting the biological evaluation of this
new tracer in vitro and in vivo for PET imaging of the anb3 integrin
receptor.
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Scheme 2 Synthesis of AmBaSar-RGD and labelled with copper-64.
8 C. L. Ferreira, D. T. Yapp, E. Lamsa, M. Gleave, C. Bensimon, P. Jurek
and G. E. Kiefer, Nucl. Med. Biol., 2008, 35, 875–882.
9 L. Wei, Y. Ye, T. J. Wadas, J. S. Lewis, M. J. Welch, S. Achilefu and C. J.
Anderson, Nucl. Med. Biol., 2009, 36, 277–285.
10 S. V. Smith, Q. J. Nucl. Med. Mol. Imaging, 2008, 52, 193–202.
11 S. V. Smith, J. Inorg. Biochem., 2004, 98, 1874–1901.
12 N. DiBartolo, A. M. Sargeson and S. V. Smith, Org. Biomol. Chem.,
2006, 4, 3350–3357.
13 S. D. Voss, S. V. Smith, N. DiBartolo, L. J. McIntosh, E. M. Cyr,
A. A. Bonab, E. A. Carter, A. J. Fischman, S. T. Treves, S. D. Gillies,
A. M. Sargeson, J. S. Huston and A. B. Packard, Proc. Natl. Acad. Sci.
U. S. A., 2007, 104, 17489–17493.
14 A. M. Sargeson, Coord. Chem. Rev., 1996, 151, 89–114.
15 P. S. Donnelly, J. M. Harrowfield, B. W. Skelton and A. H. White, Inorg.
Chem., 2000, 39, 5817–5830.
Conclusions
A novel versatile Sar caged-like bifunctional chelator AmBaSar
containing a hexa-aza cage for 64Cu radiolabelling and an aromatic
linker with carboxylic acid group for conjugate cyclic RGD has
been designed and synthesized using a conventional synthetic
strategy, with characterizations by elemental analysis, MS and 1H-
NMR. AmBaSar conjugated cyclic peptide RGD was prepared
in 80% yield, and then labelled with 64Cu with more than 80%
radiochemical yield and 95% radiochemical purity, to form a new
PET probe 64Cu-AmBaSar-RGD for imaging the anb3 integrin
receptor.
16 J. Fichna and A. Janecka, Bioconjugate Chem., 2003, 14, 3–17.
17 A. J. Beer and M. Schwaiger, Cancer Metastasis Rev., 2008, 27, 631–
644.
18 W. Cai and X. Chen, J. Nucl. Med., 2008, 49, 113S–128S.
19 R. Haubner and H. J. Wester, Curr. Pharm. Des., 2004, 10, 1439–1455.
20 R. J. Geue, T. W. Hambley, J. M. Harrowfield and A. M. Sargeson,
J. Am. Chem. Soc., 1984, 106, 5478–5488.
21 G. A. Bottomley, I. J. Clark, I. I. Creaser, L. M. Engelhardt, R. J. Geue,
K. S. Hagen, J. M. Harrowfield, G. A. Lawrance, P. A. Lay, A. M.
Sargeson, A. J. See, B. W. Skelton, A. H. White and F. R. Wilner,
Aust. J. Chem., 1994, 47, 143–179.
Acknowledgements
This work was supported by the Department of Radiology of
USC and the USC Biomedical Imaging Science Initiative. We are
grateful to Mr Allan Kershaw for assisting with the H-NMR
spectra analysis, and Nick Mordwinkin and Timothy Gallaher for
the assistance with the MS analysis.
1
22 S. Burnet, M. H. Choi, P. S. Donnelly, J. M. Harrowfield, I. Ivanova,
S. J. Jeong, Y. Kim, M. Mocerino, B. W. Skelton, A. H. White, C. C.
Williams and Z. L. Zeng, Eur. J. Inorg. Chem., 2001, 1869–1881.
23 G. C. Yeh, A. M. Beatty and J. K. Bashkin, Inorg. Chem., 1996, 35,
3828–3835.
24 A. F. Abdel-Magid, K. G. Carson, B. D. Harris, C. A. Maryanoff and
R. D. Shah, J. Org. Chem., 1996, 61, 3849–3862.
25 N. DiBartolo, A. M. Sargeson, T. M. Donlevy and S. V. Smith, J. Chem.
Soc., Dalton Trans., 2001, 2303–2309.
26 K. L. Bennett, S. V. Smith, R. M. Lambrecht, R. J. W. Truscott and
M. M. Sheil, Bioconjugate Chem., 1996, 7, 16–22.
27 M. R. Lewis, J. Y. Kao, A. L. J. Anderson, J. E. Shively and A.
Raubitschek, Bioconjugate Chem., 2001, 12, 320–324.
28 X. Chen, R. Park, M. Tohme, A. H. Shahinian, J. R. Bading and P. S.
Conti, Bioconjugate Chem., 2004, 15, 41–49.
29 T. J. Wadas and C. J. Anderson, Nature Protocols., 2007, 1, 3062–3068.
Notes and references
1 P. A. Schubiger, L. Lehmann and M. Friebe, PET Chemistry: The
Driving Force in Molecular Imaging, Springer, New York, 2006.
2 T. F. Massoud and S. S. Gambhir, Genes Dev., 2003, 17, 545–580.
3 M. J. Welch and C. S. Redvanly, Handbook of Radiopharmaceuticals:
Radiochemistry and Applications, Wiley, West Sussex, England, 2003.
4 C. H. Manning, A. Lander, E. McKinley and N. J. Mutic, J. Nucl. Med.,
2008, 49, 1401–1404.
5 M. W. Brechbiel, Q. J. Nucl. Med. Mol. Imaging, 2008, 52, 166–
173.
6 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson, Curr.
Pharm. Des., 2007, 13, 3–16.
7 S. Juran, M. Walther, H. Stephan, R. Bergmann, J. Steinbach, W. Kraus,
F. Emmerling and P. Comba, Bioconjugate Chem., 2009, 20, 347–359.
5400 | Dalton Trans., 2009, 5395–5400
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©