1-(5-Oxo-6-aza-9-aminononyl)-1,4,7,10-tetraazacyclododecan-
4,7,10-triacetic acid (6)
References
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26.
Pd (10%) on carbon (100 mg) was added to a solution
of 1-(5,11-dioxo-6,10-diaza-12-oxa-13-phenyltridecyl)-1,4,7,10-
tetraazacyclododecan-4,7,10-triacetic acid (5) (2 g, 3.14 mmol)
in MeOH (10 ml) and the suspension was stirred over 3 h under
a hydrogen atmosphere at room temperature. The mixture was
filtered over Millipore FH 0.45 lm and evaporated to give a solid
(1,68 g).
1H NMR (300 MHz, CD3OD): d 1.84 (m, 4H), 2.05 (m, 2H), 2.40
(m, 2H), 2.90–4.10 (b m, 28H). 13C NMR (300 MHz, CD3OD): d
23.80, 25.28, 28.38, 35.97, 37.24, 38.59, 50.59, 51.44, 51.81, 55.57,
57.33, 57.75, 170.11, 172.78, 174.98, 176.18. MS [M + H]+ calcd
for C22H42N6O7: 502.61, found: 503.1.
1-[5,11-Dioxo-6,10-diaza-13-(2-pyridyldithio)tridecyl]-1,4,7,10-
tetraazacyclododecan-4,7,10-triacetic acid or DO3AS-Act (7)
SPDP (620 mg,
2
mmol) was added in
a
solution
of 1-(5-oxo-6-aza-9-aminononyl)-1,4,7,10-tetraazacyclododecan-
4,7,10-triacetic acid (6) (1 g, 2 mmol) in 10 mM phosphate
buffered saline (185 ml). After 2 days, the mixture was acidified
to pH 2.5 with 3 N HCl and purified by chromatography on
R
an Amberliteꢀ XAD 1600 resin with a water–MeOH gradient.
Fractions containing the product were combined, evaporated and
freeze-dried (350 mg).
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15 G. T. Hermanson, Bioconjugate techniques, Academic Press, London,
UK, 1996.
1H NMR (600 MHz, H2O/D2O 90%): d 1.65 (o m, 6H), 2.26
(m, 2H), 2.62 (t, 2H), 2.98–3.58 (b m, 4H), 7.30 (m, 1H), 7.83 (m,
2H), 7.99 (m, 2H), 8.39 (m, 1H). 13C NMR (300 MHz, CD3OD): d
24.57, 24.93, 31.00, 36.33, 37.00, 38.81, 53.34, 55.65, 58.30, 59.16,
122.01, 123.28, 140.07, 151.27, 162.08, 174.16, 176.50. MS [M +
H]+ calcd for C30H49N7O8S2: 699.88, found: 700.2.
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Complexation of DO3AS-Act with gadolinium(III) (8)
The complexation was performed with GdCl3 in aqueous solution
at pH 7. The metal in excess was precipitated as the hydroxide
at pH 9 and filtered off over Millipore FH 0.45 lm. The mixture
was neutralized to pH 7 by adding aliquots of the free ligand
and HCl22,30 and freeze-dried. Stock solutions of the Gd-DO3AS-
Act complex in water or in 20 mM TBS buffer pH 7.2 were
prepared and used within a couple of days. The concentration
of the complex in these solutions was verified by the magnetic
susceptibility difference method23 (responsive to the paramagnetic
ion concentration) or by UV-vis spectrophotometry (responsive
to the ligand concentration). According to the latter method,
dilutions of the Gd-DO3AS-Act stock solutions were treated with
5 : 1 excess DTT at pH 7.2 to cleave the disulfide bridge in the
complex. This releases pyridine-2-thione, that has a characteristic
maximum absorbance at k = 343 nm with a molar extinction
coefficient of 8.08 103 M−1 cm−1.
22 S. Geninatti-Crich, C. Cabella, A. Barge, S. Belfiore, C. Ghirelli, L.
Lattuada, S. Lanzardo, A. Mortillaro, L. Tei, M. Visigalli, G. Forni
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Acknowledgements
Financial support from M.I.U.R. (Italian Ministry for Education)
is gratefully acknowledged (Program FIRB 2003 - “Fondo per gli
Investimenti della Ricerca di Base”, grant no. RBNE03B8KK).
The author gratefully acknowledges Prof. E. Terreno for helpful
discussions.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 4980–4987 | 4987
©