Synthetic strategy to prepare DOTA-based bifunctional chelating agent ready to bind biomolecular probes

Article abstract of DOI:10.1007/s10989-013-9348-x

Due to the continued interest in new bifunctional chelating agents (BFCA), we focused on the development of a convenient synthesis of 1,4,7,10- tetraazacyclododecane-1,4,7-tris(acetic acid)-10-butyrate mono (N-hydroxysuccinimidyl ester). It consists in the macrocycle DO3A derivatized with an aliphatic linker containing an active ester that requires selective and mild conditions to react with the targeting biomolecule. It is important to underlay the versatility of the obtained BFCA, which can be conjugated both to a biomolecule (protein, Fab fragment) or to a synthetic molecule. In a subsequent step, the developed chelator was successfully conjugated to a peptide sequence.

Full text of DOI:10.1007/s10989-013-9348-x

Int J Pept Res Ther (2013) 19:199–202
DOI 10.1007/s10989-013-9348-x
Synthetic Strategy to Prepare DOTA-Based Bifunctional
Chelating Agent Ready to Bind Biomolecular Probes
•
Enrica Calce Luca Monfregola Stefania De Luca
•
Accepted: 15 April 2013 / Published online: 25 April 2013
Ó Springer Science+Business Media New York 2013
Abstract Due to the continued interest in new bifunctional
chelating agents (BFCA), we focused on the development of
a convenient synthesis of 1,4,7,10-tetraazacyclododecane-
1,4,7-tris(acetic acid)-10-butyrate mono (N-hydroxysucc-
inimidyl ester). It consists in the macrocycle DO3A deriva-
tized with an aliphatic linker containing an active ester that
requires selective and mild conditions to react with the tar-
geting biomolecule. It is important to underlay the versatility
of the obtained BFCA, which can be conjugated both to a
biomolecule (protein, Fab fragment) or to a synthetic mol-
ecule. In a subsequent step, the developed chelator was
successfully conjugated to a peptide sequence.
Introduction of a reactive group into this macrocycle
allows the preparation of the so-called bifunctional che-
lating agents which are commonly used to label biomole-
cules with radiometal or paramagnetic ions for biomedical
applications (Lattuada et al. 2011). Many examples of
DOTA-like bifunctional chelators have been published (Li
et al. 2009; Jamous et al. 2012), most of them were
obtained as activated esters of DOTA in order to react with
an amino group, usually present in an antibody or peptide,
to form an amide bond. Among the active esters, for
instance, one of the most commonly prepared derived from
N-hydroxysuccinimide (Mier et al. 2005).
DO3A-tris-tert-butyl ester is widely employed as a
precursor to explore new protocols of synthetically useful
DOTA-based BFCA, in fact the free secondary amino
group can be derivatized in order to conjugate the macro-
cycle to a target-specific biomolecules (Yoo and Pagel
2006; Bernhard et al. 2012; Barge et al. 2009; Wangler
et al. 2008; Mishra and Chatal 2001; Knor et al. 2007).
In addition, it is characterized by an accessible price, so
that we decided to develop a convenient protocol to prepare
a bifunctional chelating agent containing the ligand DO3A
and a reactive group allowing the conjugation to a bio-
logical probe. An aliphatic spacer is present between the
two moieties, in order to avoid that the bulky macrocycle
could affect the interaction with the biological target.
Keywords DOTA ꢀ Bifunctional chelating agent ꢀ
Biological probe ꢀ Peptide labeling
Introduction
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic acid) belongs to the family of cyclic chelating agents
able to form metal ions complexes endowed with thermo-
dynamic stability and kinetic inertness, for applications
both as diagnostic and therapeutic agent. Indeed, this
macrocycle is very useful for the complexation of b--
emitting radionuclides, like 90Y, 177Lu, widely employed in
nuclear medicine protocols, and trivalent ions like Gd(III)
for application in magnetic resonance imaging as contrast
agent (Dischino et al. 1991; Mishra et al. 2006).
Materials and Methods
DO3A-tris-tert-butyl ester was purchased from Macrocyc-
lics (Dallas, TX); N-hydroxysuccinimide, 4-bromobutyric
acid, potassium carbonate and N,N0-dicyclohexylcarbodi-
imide were purchased from Sigma-Aldrich (St Louis, MO).
Fmoc protected amino acids, N-hydroxybenzotriazole
E. Calce ꢀ L. Monfregola ꢀ S. De Luca (&)
Institute of Biostructure and Bioimaging, National Research
Council, Via Mezzocannone 16, 80138 Naples, Italy
e-mail: stefania.deluca@cnr.it
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OH
N
(HOBT), benzotriazol-1-yl-oxy-tris-pyrrolidino-phospho-
nium (PyBOP) were purchased from Calbiochem-Nova-
biochem (Laufelfingen, Switzerland), piperidine and
diisopropylethylamine (DIPEA) were purchased from Fluka
(Milwaukee, WI), Rink Amide MBHA resin and all
remaining solvents were purchased from Aldrich (St Louis,
MO) or Fluka (Milwaukee, WI) and were used without fur-
ther purification, unless otherwise stated.
O
O
O
O
O
O
N
Br
Br
OH
O
O
i
1
tBu
O
OtBu
N
N
N
ii
O
Analytical RP-HPLC runs were carried out on a HP
Agilent Series 1100 apparatus using a Phenomenex (Tor-
rance, CA) C18 column, 4.6 9 250 mm with a flow rate of
1.0 mL min-1. Preparative RP-HPLC was carried out on a
Shimadzu 8A apparatus equipped with an UV Shimadzu
detector using a Phenomenex (Torrance, CA) C18 column,
22 9 250 mm with a flow rate of 20 mL min-1. The sys-
tem solvent used was H2O 0.1 % TFA (A) and CH3CN
0.1 % TFA (B), with a linear gradient from 5 to 70 % B in
30 min. LC-ES-MS data were obtained using a Finnigan
Surveyor MSQ single quadrupole electrospray ionisation
mass spectrometer coupled with a Finnigan Surveyor
HPLC (Finnigan/Thermo Electron Corporation San Jose,
CA, USA).
O
HN
tBu
O
O
tBu
O
OtBu
N
N
N
N
O
O
O
O
N
O
tBu
O
O
2
iii
HO
O
OH
N
N
O
O
O
1H NMR spectra were recorded on a Varian Unity Inova
400 MHz spectrometer equipped with z-axis pulsed-field
gradients and a triple resonance probe. Samples were
prepared in tubes with a diameter of 5 mm using 0.5 mL of
deuterated solvent (CD3OD). The experiments were
acquired at 25 °C and the spectra were recorded with 256
scans and a relaxation delay of 1 s.
O
N
N
N
O
HO
O
3
Scheme 1 Synthesis of DO3A based-bifunctional chelator. Reagents
and conditions: (i) DCC (1 equiv.), DCM, 0 °C, 4 h; (ii) K2CO3 (1
equiv.), DMF, 6 h, rt; (iii) TFA/TIS/EDT (96.5:1:2.5), 3 h
O
O
Synthesis
H
N
H
N
H2N
N
O
H
O
O
Synthesis of 4-bromobutyric acid N-hydroxysuccinimidyl
ester (1). 4-bromobutyric acid (100 mg, 0.6 mmol) was
dissolved in dry dichloromethane (anhydrous, C99.8 %,
2 mL) with 1 equiv. of N-hydroxysuccinimide (69 mg,
0.6 mmol). N,N0-Dicyclohexylcarbodiimide (1 equiv.,
124 mg), previously dissolved in the minimum volume of
the same solvent, was added dropwise. The solution was
cooled in an ice-water bath and stirred vigorously for 4 h.
The white solid N,N0-dicyclohexylurea was removed by
filtration and the solvent was evaporated in vacuo to give
the final product 1, as assessed by analytical HPLC and
mass spectrometry analysis. (Yield 80 %). ES-MS: calcd.
[M?H]?, 265.1; found, m/z 265.1.
S-Trt
i, ii
HO
O
OH
N
N
N
N
O
O
O
O
O
H
H
N
N
NH2
N
H
N
HO
H
O
O
SH
Scheme 2 Solid phase synthesis of DO3A-peptide conjugate.
Reagents and conditions: (i) 1,4,7,10-Tetraazacyclododecane-1,4,7-
tris (tert-butyl acetate)-10-butyrate mono (N-hydroxysuccinimidyl
ester) 2 (1.5 equiv.), DIPEA (3 equiv.), DMF, 1 h; (ii) TFA/TIS/EDT/
H20 (93.5:2:2.5:2), 3 h
1H NMR: d (ppm) 2.28 (BrCH2CH2CH2COO(NHS), p,
J = 6.37, 6.78 Hz, 2H); 2.85 (BrCH2CH2CH2COO(NHS),
t, 2H); 2.87 (CH2 NHS, broad signal, 4H); 3.59 (BrCH2CH
2CH2COO(NHS), t, J = 6.49 Hz, 2H).
DO3A-tris-tert-butyl ester dissolved in 2 mL of DMF and
in presence of 1 equiv. of K2CO3 (5 mg, 0.04 mmol). The
mixture was stirred at room temperature for 6 h and the
course of the alkylation reaction was followed by HPLC-
ES-MS analysis. The mixture was concentrated under
Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-tris
(tert-butyl acetate)-10-butyrate mono (N-hydroxysuccin-
imidyl ester) (2). 1.2 equiv. of 1 (13 mg, 0.05 mmol)
were placed in a round-bottom flask containing 20 mg of
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Int J Pept Res Ther (2013) 19:199–202
201
vacuum and the crude product was purified by RP-HPLC to
be fully characterized by mass spectrometry. (Yield 50 %).
ES-MS: calcd. [M?H]?, 698.9; found, m/z 698.4.
1H NMR: d (ppm) 1.51 (tert-butyl CH3), broad signal,
27H); 2.00 (NCH2CH2CH2COO(NHS), p, t, J = 6.50,
6.26 Hz, 2H); 2.77 (NCH2CH2CH2COO(NHS), t, J =
6.97 Hz, 2H); 2.69 (CH2 NHS, broad signal, 4H), 4.13
(NCH2CH2CH2COO(NHS), t, J = 5.83 Hz, 2H); 2.93,
3.06, 3.20, 3.30, 3.51, 3.63, 3.72, 3.98 (broad signals,
NCH2 DOTA, 22H).
resin (0.28 mmol g-1 substitution) was used as solid sup-
port, as it releases peptide amidated at C-terminus upon
acid treatment. All amino acid couplings were performed
twice for 20 min by using an excess (4 equiv.) of each
amino acid derivative. The amino acids were activated
in situ by the standard HOBt/PyBOP/DIPEA protocol.
Fmoc deprotection was performed with 20 % piperidine in
DMF. The last coupling was performed using compound 2
(1.5 equiv.) and DIPEA (3 equiv.) in DMF. After 1 h the
reaction was monitored by Kaiser test (Kaiser et al. 1970).
The peptide cleavage from the solid support and the
simultaneous removal of all protecting groups from the
amino acid residues and from the BFCA was carried out by
suspending the fully protected compound-resin in TFA/
TIS/EDT/H2O/(93.5:2:2.5:2) for 3 h followed by filtration.
The solution was then concentrated and the crude product
isolated by precipitation into cold diethyl ether. RP-HPLC
and mass spectrometry analysis confirmed the presence of
the desired compound which was isolated by HPLC pre-
parative (yield 25 %). HPLC: tR = 11.1; ES-MS: calcd.
[M?H]?, 761.9; found, m/z 762.4
Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-
acetic acid 10-butyrate mono (N-hydroxysuccinimidyl
ester) (3). 10 mg of compound 2 was treated with 200 lL
of a solution of TFA/EDT/TIS (Trifluoroacetic acid/Trii-
sopropylsilane/Ethanedithiol) (96.5:2.5:1) for 3 h. Then the
solution was concentrated and the product 3 was isolated
by precipitation into cold diethyl ether to be fully charac-
terized by mass spectrometry. (Yield 85 %). ES-MS: calcd.
[M?H]?, 530.6; found, m/z 530.1.
1H NMR: d (ppm) 2.01 (NCH2CH2CH2COO(NHS), p,
J = 6.02, 6.30 Hz, 2H); 2.73 (NCH2CH2CH2COO(NHS),
t, J = 7.16 Hz, 2H); 2.69 (CH2 NHS, broad signal, 4H);
4.13 (NCH2CH2CH2COO(NHS), t, J = 5.80 Hz, 2H);
2.96, 3.05, 3.15, 3.31, 3.54, 3.57, 3.73, 3.98 ppm (broad
signals, NCH2 DOTA, 22H).
Results and Discussion
Synthesis of DO3A-peptide conjugate. Peptide synthesis
was carried out by solid phase method using the standard
Fmoc procedure. Appropriate Fmoc-amino acid derivatives
were employed (Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-
Cys(Trt)-OH, Fmoc-Gly-OH) and a Rink Amide MBHA
A practical and easy synthetic protocol in order to prepare
in good yield a versatile bifunctional chelating agent was
developed. As shown in Scheme 1, the first synthetic step
consisted in activating the carboxylic function of the
4-bromobutyric acid with NHS (N-hydroxysuccinimide) in
Fig. 1 LC–MS profile of the DO3A-peptide conjugate crude product
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References
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presence of the inorganic base (K2CO3), the stirring was
kept for 6 h. The amount of K2CO3 should not exceed 1
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In conclusion, starting from inexpensive and readily
available materials, we have developed a fast and
straightforward procedure to synthesize useful BFCA
which can be conjugated to biological ligands, in order to
label them with metal ions (radionuclides or paramagnetic
metal) for targeted diagnostic imaging or therapy.
Acknowledgments We thank Mr. Leopoldo Zona (National
Research Council, Naples) for NMR technical assistance.
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