Synthesis and properties of a neutral derivative of diethylenetriaminepentaacetic acid (DTPA)

Article abstract of DOI:10.1016/j.bmcl.2006.06.088

A neutral bifunctional derivative of diethylenetriaminepentaacetic acid europium(III) (11) was synthesized and its suitability to dissociation-enhanced lanthanide fluorescence immunoassay was investigated.

Full text of DOI:10.1016/j.bmcl.2006.06.088

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4760–4762
Synthesis and properties of a neutral derivative of
diethylenetriaminepentaacetic acid (DTPA)
Jari Peuralahti, Liisa Merio¨, Veli-Matti Mukkala, Kaj Blomberg and Jari Hovinen*
PerkinElmer Life and Analytical Sciences, Turku Site, PO Box 10, FIN-20101 Turku, Finland
Received 16 June 2006; revised 29 June 2006; accepted 29 June 2006
Available online 17 July 2006
Abstract—A neutral bifunctional derivative of diethylenetriaminepentaacetic acid europium(III) (11) was synthesized and its suit-
ability to dissociation-enhanced lanthanide fluorescence immunoassay was investigated.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Because of its excellent metal-chelating properties
diethylenetriaminepentaacetic acid (DTPA) is one of the
most widely used organic ligands in magnetic resonance
imaging (MRI) and positron emission tomography
(PET).1–3 Indeed, the first FDA-approved contrast agent
in clinical use is the Gd3+ DTPA chelate.4 The corre-
sponding 111In and 68Ga chelates, in turn, are suitable
for PET applications,5 while Eu3+, Tb3+, Sm3+, and
Dy3+ chelates can be used in applications based on disso-
ciation-enhanced lanthanide fluorescence immunoassay
(DELFIA).6 99mTc, in turn, is suitable for single positron
emission computed tomography (SPECT).7,8 Bioactive
molecules labeled with 111In or 117mSn DTPA may find
applications as target-specific radiopharmaceuticals.9
tact blood–brain barrier. Naturally, bioactive molecules
labeled with this type of chelates have lower cell perme-
ability than the corresponding intact molecules.12 This
diminishes the suitability of DTPA chelates to in vivo
applications. Furthermore, the negatively charged che-
lates may bind unselectively to positively charged bind-
ing sites of target molecules, such as antibodies, via
electrostatic interactions which may result in low recov-
eries.13 Naturally, all these above-mentioned problems
will be even more serious when the target molecule is
labeled with several charged chelates.14
Several of the above-mentioned problems can be avoided
by neutralizing the net charge of the chelate by substitut-
ing two of the DTPA acetates with carboxamido func-
tions. Indeed, several this type of chelators have been
synthesized15,16 and one of them, Gd[DTPA-bis(ethyla-
mide)]17 (gadodiamide; Omniscan), is currently in clinical
use. However, it has been shown that if one of the acetic
acid groups of DTPA is used for conjugation, the result-
ing chelateis less stable than the parent DTPA molecule.18
This may be a serious problem especially in in vivo appli-
cations if toxic metal ions have to be used.
In several applications, covalent conjugation of DTPA
to bioactive molecules is required. This can be per-
formed in solution by allowing an amino or mercapto
group of a bioactive molecule to react with isothiocy-
anato, haloacetyl or 3,5-dichloro-2,4,6-triazinyl deriva-
tives of the label molecules.10 Several bifunctional
DTPA derivatives are currently commercially available.
Also a solid-phase method for the introduction of
DTPA to synthetic oligonucleotides and oligopeptides
has been demonstrated.11
We present here synthesis of neutral derivatives of
DTPA chelate which allow covalent conjugation of bio-
active molecules. Also their suitability to DELFIA-
based assays is demonstrated.
The net charge of DTPA chelates is most commonly À2,
which may cause problems in several applications. The
most commonly used MRI contrast agent Gd-DTPA
(Magnevist) distributes throughout the extracellular
and intravascular fluid spaces, but does not cross an in-
Synthesis of the neutral DTPA chelate is depicted in
Scheme 1. Initially, 2-amino-N-(2-aminoethyl)-3-(4-
nitrophenylphenyl)propanamide19 (1) was converted to
the Schiff base, 2, by treatment with benzaldehyde, bor-
ane reduction of which yielded the amine 3. It was then
alkylated with tert-butyl bromoacetate to give 4 in good
Keywords: DTPA; Europium; DELFIA; Imaging.
*
Corresponding author. E-mail: jari.hovinen@perkinelmer.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.088

J. Peuralahti et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4760–4762
4761
Scheme 1. Reagents and conditions: (i) PhCHO in EtOH; (ii) a—BH3 Æ THF, o/n at reflux, b—concd HCl, 3 h at reflux, c—aq ammonia, d—
purification on Al2O3; (iii) BrCH2COO-t-Bu, DIPEA in DMF, o/n at rt; purification on silica gel (79%); (iv) Pd/C, NaBH4, in MeOH, 30 min at rt;
purification on silica gel (58%); (v) Boc2O, TEA, in MeCN, 2 h at rt, purification on silica gel (92%); (vi) Pd/C, ammonium formate, in MeOH, 15 min
at reflux; purification on silica gel (75%); (vii) iodoacetamide, K2CO3 in MeCN, 2 h at reflux, purification on silica gel (86%); (viii) TFA 4 h at rt; (ix)
EuCl3; pH 6–7; 2 h at rt; (x) thiophosgene, CHCl3, aq NaHCO3, 1 h at rt.
yield. Selective reduction of the nitro group to the amino
function using the mixture of Pd/C and NaBH4 in meth-
anol20 gave rise to 5. After protection of the aromatic
amino group as tert-butyl carbamate, the benzyl groups
were removed by hydrogenation with Pd/C in the pres-
ence of ammonium formate.21 The secondary amines
of 7 were alkylated with iodoacetamide to give the pro-
tected ligand 8. After cleavage of the protecting groups
by acidolysis, the free ligand 9 was converted to the cor-
responding europium(III) chelate 10 by treatment with
europium(III) chloride.22 Finally, reaction of 10 with
thiophosgene22 gave the activated chelate 11.
To study the applicability of neutral DTPA derivatives
to DELFIA assays, the stability of 10 and the corre-
sponding charged chelate 12 in DELFIA Enhancement
Solution and Inducer was compared. Accordingly, the
stabilities of 10 and 12 was equal, giving times needed
for complete dissociation <5 and 30 min for Inducer
and DELFIA Enhancement Solution, respectively.23
Scheme 2.
The assay conditions were optimized for each tracer
individually, and the analytical sensitivities of the opti-
mized standard curves were defined. The correlation be-
tween the methods was studied with a small sample
panel. The on-board stability was tested up to one week
in instrument-like conditions. Also the sensitivity to the
Next, comparison of the performance of the tracer 15
and the corresponding DTPA derivative 16 to tracer
used in AutoDELFIA Neonatal T4 (thyroxine) kit 17 was
performed. Tracer synthesis is outlined in Scheme 2.24

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J. Peuralahti et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4760–4762
Table 1. Comparison of the performance of the tracer 15 and the corresponding DTPA derivative 16–17
Tracer
15
16
17
Analytical sensitivitya
0.42 lL/dL
0.35 lL/dL
0.63 lL/dL
Correlation to AutoDELFIA neonatal T4 assay
Mean bias
Interference with EDTAb
y = 1.02 · À0.37, R = 0.87, n = 27
y = 1.2 · À3.22, R = 0.94, n = 27
À0.7%
À0.1%
No
No
Yes
a Analytical sensitivity was detected as 2 SD below the mean of the zero standard measurement value.
b Tested with EDTA concentration up to 12 mg/mL.
9. Volkert, W. A.; Hoffman, T. J. Chem. Rev. 1999, 99,
2269.
interference of EDTA-containing samples was studied.
The results are summarized in Table 1.
10. Fichna, J.; Janecka, A. Bioconjugate Chem. 2003, 14, 3.
11. Peuralahti, J.; Jaakkola, L.; Mukkala, V.-M.; Hovinen, J.
Bioconjugate Chem. 2006, 17, 855.
12. Rogers, B. E.; Anderson, C. J.; Connett, J. M.; Guo, L.
W.; Edwards, W. B.; Sherman, E. L.; Zinn, K. R.; Welch,
M. J. Bioconjugate Chem. 1996, 7, 511.
The shapes of the calibration curves obtained with opti-
mized amounts of tracer and antiserum were slightly dif-
ferent with the three tracers. All tracers were sensitive
enough at clinically important range. Assays with the
tested tracers compared well to the AutoDELFIA
Neonatal T4 assay and no significant level differences
were obtained. In strict contrast to tracer 17, neither
15 nor 16 was sensitive to EDTA. Also their stabilities
under acidic conditions were similar.
13. Rosendale, B. E.; Jarrett, D. B. Clin. Chem. 1985, 31,
1965.
14. Peuralahti, J.; Suonpa¨a¨, K.; Blomberg, K.; Mukkala,
V.-M.; Hovinen, J. Bioconjugate Chem. 2004, 15, 927.
15. Hanaoka, K.; Kikuchi, K.; Urano, Y.; Narazaki, M.;
Yokawa, T.; Sakamoto, S.; Yamaguchi, K.; Nagano, T.
Chem. Biol. 2002, 9, 1027.
16. Feng, J.; Sun, G.; Pei, F.; Liu, M. Bioorg. Med. Chem.
2003, 11, 3359.
17. Konings, M. S.; Dow, W. C.; Love, D. B.; Raymond, K.
N.; Quay, S. C.; Rocklage, S. M. Inorg. Chem. 1990, 29,
1488.
Accordingly, two of the DTPA acetates can be substitut-
ed with carboxamido functions without loss of the
desired chelate stability. The neutral DTPA chelates
synthesized here are as stable as the corresponding
charged derivatives. Since the preliminary results are
promising we are currently synthesizing tracers labeled
with several neutral DTPA derivatives.
18. Paul-Roth, C.; Raymond, K. N. Inorg. Chem. 1995, 34,
1408.
19. Corson, D. T.; Meares, C. F. Bioconjugate Chem. 2000, 11,
292.
20. Peuralahti, J.; Hakala, H.; Mukkala, V.-M.; Hurskainen,
P.; Mulari, O.; Hovinen, J. Bioconjugate Chem. 2002, 13,
870.
21. Ram, S.; Spicer, L. D. Synth. Commun. 1987, 17, 415.
22. Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.;
Hemmila¨, I. Bioconjugate Chem. 1994, 5, 278.
23. The chelates (ca. 1 mg) were dissolved either in Inducer or
Enhanchement Solution (PerkinElmer), and the dissocia-
tion of europium at 25 ꢁC was followed using a time-
resolved fluorometer (Victor2V).
24. Compound 14 was synthesized by allowing L-thyroxine to
react with Fmoc-aminohexanoic acid N-hydroxysuccinate
(1.1 equiv) in dry DMF in dark (1 h at rt). Piperidine was
then added (removal of Fmoc group), and the reaction
was allowed to proceed for 30 min at rt before being
concentrated in vacuo. The title compound was obtained
by precipitation from methanol.
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