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48.9 (CH2), 68.1 (CH2), 69.4 (CH2), 69.7 (CH2), 71.1 (CH2), 71.3
(CH2), 116.1 (CH), 122.2 (CH), 148.3, 169.0 ppm. TLC: Rf = 0.8
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+ Na]+ 475.2062; found 475.2060.
General Procedure for the Synthesis of Eu-1–4: A degassed aqueous
solution of EuCl2 (1 equiv.) was mixed with a degassed aqueous solu-
tion of a cryptand (2 equiv.). The resulting mixture was stirred for
12 h at ambient temperature under Ar. Degassed PBS (10ϫ) was
added, and stirring was continued for 30 min. The concentration of
Eu in the resulting solution was verified by ICP-MS, and the solution
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pH Buffers: The following commercially available buffers were de-
gassed and used in relaxometric experiments: glycine/HCl (pH = 3),
acetate (pH = 5), PBS (pH = 7.4), 2-amino-2-(hydroxymethyl)prop-
ane-1,3-diol (TRIS) (pH = 8), and glycine/NaOH (pH = 10).
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cryptate (5 mm) was prepared in degassed PBS. To an aliquot of this
solution was added a solution of Ca2+ (12.1 mm) in degassed PBS
[12] A xylenol orange-based colorimetric assay was used to test for
the presence of free europium as described in ref.[14] When a solu-
tion of EuCl2 (2.5 mm) in PBS was prepared, a precipitate imme-
diately formed. The filtrate of the mixture was exposed to air to
allow oxidation of Eu2+ to Eu3+, and the Eu3+ concentration was
measured with xylenol orange as an indicator using a calibration
curve made from a purchased Eu standard. A concentration of
1.63 μm was measured, which corresponds to 0.07% of the origi-
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such that the resulting solution was 2.5 mm in both Eu2+ and Ca2+
.
This solution was stirred at 37 °C under Ar. Aliquots were taken at
90, 180, 420, 1500, 3300, and 4740 min after the addition of Ca2+
.
All aliquots were filtered using 0.2 μm filters prior to T1 measure-
ments. The t1 value of these aliquots (60 MHz, 37 °C) was immedi-
ately measured at each time point. The experiment was triplicated
with independently prepared solutions. The entire procedure was re-
peated using Mg2+ (16.4 mm) and Zn2+ (9.97 mm) in place of Ca2+
(12.1 mm). Stastical analysis of variance was performed using the pro-
gram found at faculty.vassar.edu/lowry/anova1u.html.
Supporting Information (see footnote on the first page of this article):
1H and 13C NMR spectra of 3 and 4.
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This research was supported by startup funds from Wayne State Uni-
versity (WSU) and by the National Institute of Biomedical Imaging
and Bioengineering of the National Institutes of Health (Pathway
to Independence Career Transition Award, R00EB007129). J. G. was
supported by a Paul and Carol Schaap Graduate Fellowship, and
M. J. A. gratefully acknowledges a Schaap Faculty Scholar Award.
We thank Latif Zahid and Yimin Shen for performing imaging ex-
periments.
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Received: October 21, 2011
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Published Online: February 8, 2012
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