Amphiphilic EuDOTA-tetraamide complexes form micelles with enhanced CEST sensitivity

Article abstract of DOI:10.1002/ejic.201101369

The synthesis and characterization of four new DOTA-tetraamide ligands having variable alkyl chain lengths (C₁, C₁₂, C ₁₄, and C₁₆) and their respective europium(III) complexes are reported. The three EuL comple

Full text of DOI:10.1002/ejic.201101369

FULL PAPER
DOI: 10.1002/ejic.201101369
Amphiphilic EuDOTA-Tetraamide Complexes Form Micelles with Enhanced
CEST Sensitivity
Osasere M. Evbuomwan,[a] Garry Kiefer,[a,b] and A. Dean Sherry*[a,c]
Keywords: Imaging agents / Europium / Micelles
The synthesis and characterization of four new DOTA-
tetraamide ligands having variable alkyl chain lengths (C1,
at or near room temperature. The water residence lifetimes
obtained by fitting the CEST spectra to the Bloch equations
increased in parallel with an increase in alkyl carbon chain-
length. By comparisons with the monomethylamide complex,
which served as control, the data illustrate that micelle for-
mation serves to slow the rate of water exchange in these
systems. The complex having the largest CEST effect per
unit EuIII concentration (the C16 analog) had a detection limit
C12, C14, and C16) and their respective europium(III) com-
plexes are reported. The three EuL complexes having long
alkyl chains spontaneously form micelles of variable size.
The critical micelle concentration differed for each complex
(lower for the C16 complex than the C12 complex) while mi-
celle size increased with increasing alkyl chain length.
Chemical exchange saturation transfer (CEST) experiments
showed that all four EuIII complexes displayed slow-to-inter-
mediate water exchange kinetics. As expected, the CEST
signals in these complexes decreased with increasing tem-
peratures due to faster water exchange but, interestingly, the
CEST signals for the C14 and C16 complexes approached a
maximum near 25 °C consistent with exchange limited CEST
of 5.3 μ
in sensitivity relative to the monomethylamide control (de-
tection limit ≈ 1.3 m ). These features highlight the potential
M. This represents an approximate 250-fold increase
M
of using micelle-based systems such as these as paramag-
netic chemical exchange saturation transfer (PARACEST)
agents for molecular imaging by MRI.
image contrast by decreasing the total bulk water signal in-
tensity. In a typical CEST experiment, the resonance of the
labile protons on the CEST agent may be selectively satu-
rated by using a specific radiofrequency (RF) pulse. Satu-
rated magnetization is then transferred from the labile pro-
tons to the bulk water protons through a chemical exchange
process that has to be maintained within the slow-to-inter-
mediate regime. The result is a decrease in the bulk water
signal intensity which translates into a negative contrast in
an MR image.[2]
Early versions of CEST agents consisted of low molecu-
lar weight diamagnetic compounds with Δω values within
5 ppm of bulk water.[3,4] These small Δω values not only
restricted CEST to slow exchanging systems but also in-
creased the possibility of directly saturating the bulk water
signal. Hence, the discovery that certain paramagnetic lan-
thanide complexes with DOTA-tetraamide ligands induced
large hyperfine shifts in the bound water protons and other
proximate exchanging protons in the chelating ligand[5] ulti-
mately led to the development of a new class of CEST
agents now referred to as paramagnetic chemical exchange
saturation transfer (PARACEST) agents.
Introduction
Magnetic resonance imaging (MRI) has evolved into one
of the most powerful non-invasive techniques in diagnostic
medicine because it uses non-ionizing radiation and allows
exquisite high resolution imaging of the human anatomy.
In addition, the clinical value of MRI has been significantly
augmented by the availability of gadolinium-based T1
agents as approved contrast media for enhancing soft tissue
contrast by shortening the longitudinal relaxation times of
water protons. While these agents are very effective at high-
lighting tissues with abnormal perfusion such as most tu-
mors, current contrast agents provide no direct physiologi-
cal or metabolic information. This need has stimulated a
rich and active area of research devoted to the design of
new MRI agents capable of producing water contrast by
mechanisms other than relaxation.[1]
Chemical exchange saturation transfer (CEST) agents
represent an emerging class of contrast media that can alter
[a] Department of Chemistry, University of Texas at Dallas,
P. O. Box 830668, Richardson, Texas 75083, USA
E-mail: sherry@utdallas.edu
[b] Macrocyclics, Inc.,
1309 Record Crossing, Dallas, Texas 75235, USA
[c] Advanced Imaging Research Center, UT Southwestern Medical
Center,
PARACEST agents possess a number of appealing fea-
tures over their diamagnetic counterparts; for example, Δω
which can range from +500 ppm to –720 ppm depending
upon which lanthanide is used.[6] Eu3+ complexes of
DOTA-tetraamide ligands have been the most widely
5323 Harry Hines Boulevard, Dallas, Texas 75390, USA
E-mail: dean.sherry@utsouthwestern.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201101369.
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Micelles with Enhanced CEST Sensitivity
studied PARACEST agents to date because these com- factor for CEST are the water or proton exchange rates may
plexes display the slowest water exchange kinetics of all lan- be altered, either to an advantage or disadvantage for
thanide complexes.[7] In addition, this ligand architecture is CEST, when a PARACEST complex is part of a micelle
easily modified to create agents that are sensitive to a vari- structure. Here, we address both questions by reporting the
ety of physiological parameters,[8,9] metal ions,[10] metabo- synthesis and characterization of four new PARACEST
lites,[11,12] and enzymes.[13–15] Nevertheless, first generation agents with variable alkyl carbon chain-lengths (Scheme 1)
DIACEST and PARACEST agents are relatively insensi- and the influence of carbon chain-length on the CEST
tive, typically requiring millimolar concentrations to pro- properties of these systems.
duce a detectable signal. The development of more sensitive
PARACEST agents is critical to make them effective for
Results and Discussion
molecular imaging of such biological processes. While a
handful of sensitivity enhancement methods are possible,
the most straightforward is to increase the number of ex-
changing groups (N) per agent. Attempts at improving sen-
sitivity by increasing N have focused largely on macromo-
lecular agents (both diamagnetic and paramagnetic)[16–20]
and nanoparticulate agents.[21–24]
Synthesis
Scheme 2 outlines the synthetic procedure for preparing
the compounds used in this study. The mono-protected
macrocycle 2 was synthesized according to previously pub-
lished methods.[32] Alkylation of 2 was carried out with 3a
or 3b using standard conditions followed by purification
using silica gel column chromatography. The CBz protect-
ing group was then removed by hydrogenolysis in the pres-
ence of H2 and palladium on carbon (10%) to yield 5a and
5b, respectively. Subsequently, alkylation of 5a or 5b with
an N-alkyl-2-bromoacetamide 6a–d was performed and the
crude products purified by column chromatography. The
ethyl and tert-butyl ester groups were removed with LiOH
or TFA, respectively, to yield the target ligands. Europium
complexes with ligands 1, 12, 14 and 16 were prepared by
combining stoichiometric quantities of each ligand and eu-
ropium(III) chloride in water while maintaining the pH be-
tween 5.5 and 6. After 24 h, the complexation was complete
as confirmed by the absence of non complexed Eu3+ using
xylenol orange titration. The resulting solutions were then
lyophilized to yield the final compounds used in this study.
Based upon the crystal structure of EuDOTA-(glycine
ethyl ester)4,[5] and previous studies on a number of other
similar ligand systems,[33,34] the approximate solution geom-
etry of the EuIII complexes prepared here was predicted to
have the three glycinate arms oriented axially above the
Eu3+–OH2 bond and the single alkylamide sidearm ori-
ented equatorially (Figure 1). This model is based on the
known tendency for smaller, polar amide arms to adopt
an axial conformation due to lower steric hindrance, while
bulkier, hydrophobic pendant arms, lacking an α-carbonyl
The use of nanoparticle platforms for sensitivity en-
hancement is attractive for CEST since such systems have
the potential of delivering a high payload of exchangeable
groups, either through surface conjugation or encapsulation
of multiple small-molecule agents, to a target of interest.
While this method has proven efficient in some cases,[21,24]
other nanoparticulate systems have been shown to disrupt
the CEST signal by rapid catalysis of the proton exchange
from an encapsulated agent or by interference from a broad
magnetization transfer-like signal which obscures CEST
from paramagnetic exchange sites.[23] These results emphas-
ize the importance of choosing the proper nanoparticle
composition and the physical location of the CEST agent
in the nanoparticle. In this work, we report a series of am-
phiphilic PARACEST agents capable of assembling into
micelles as an approach toward increasing N. Micelles have
been widely studied as vehicles for drug delivery due to the
possibility of controlling their structural characteristics
through simple changes in physicochemical properties such
as size and surface charge.[25] More recently, it has been
shown that the T1 sensitivity of GdIII can be amplified by
choosing amphiphilic ligands capable of micelle formation.
The T1 enhancement in such complexes results from a com-
bination of an increase in N plus the added benefit of re-
stricted molecular motion.[26–29] Given these proven advan-
tages, a micellar platform is also attractive for PARACEST
simply on the basis of an increase in N, the number of ex-
changing protons that can be saturated and then exchange
into the large pool of bulk water. One unknown but critical
Figure 1. Hyperchem molecular model illustrating the relative sizes
of the Eu-(16) head-group and the C16 hydrophobic tail. Color
code, black: carbon, blue: nitrogen, red: oxygen, white: hydrogen,
central white: europium.
Scheme 1. General structure of PARACEST agents in this study.
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O. M. Evbuomwan, G. Kiefer, A. D. Sherry
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Scheme 2. Synthetic Scheme for the ligands and Eu3+ complexes examined in this study.
group have a preference for an equatorial position.[34] This larity of the amide pendant arms compared to the acetate
suggested that Eu-(12), Eu-(14), and Eu-(16) would likely arms and hence their overall, greater hydrophobicity. These
form micelles in solution with the paramagnetic center lo- results suggest that Eu-(12), Eu-(14), and Eu-(16) have a
cated on the micelle surface and the hydrophobic tails bur- greater chance of retaining a micelle structure in blood
ied inside the micelle core.
compared to previously reported gadolinium analogues, or
perhaps will interact more strongly with hydrophobic re-
gions of serum albumin and exhibit a longer vascular reten-
tion lifetime.[36,37]
Determination of Critical Micelle Concentration
An interesting property exhibited by surfactants is their
ability to self-assemble into micelles at concentrations above
the critical micelle concentration (CMC). To test for micelle
formation, fluorescence titrations of each complex were
performed in the presence of 8-anilino-1-naphthalenesul-
fonic acid (ANS), an anionic dye that is weakly fluorescent
in water but becomes highly fluorescent when located in a
lipid environment.[30] A large increase in the fluorescence
intensity and a blue shift in the emission spectrum of ANS
is indicative of micelle formation. Plots of fluorescence in-
tensity vs. EuL concentration showed a clear break in lin-
earity at the CMC for each species (see Supporting Infor-
mation). The CMC determined from these plots for Eu-
(12), Eu-(14), and Eu-(16) were 67 μm, 48 μm, and 26 μm,
respectively, consistent with the anticipated decrease in
CMC with increasing carbon chain length. An identical ti-
tration for Eu-(1) did not show a break or increase in fluo-
Dynamic Light Scattering
Micelle size, as determined by dynamic light scattering
(DLS) at two concentrations, was found to increase mod-
estly as the alkyl carbon chain length increased from 12 to
16 carbons (3.7–5.5 nm, Figure 2). These diameters agreed
closely to those estimated from simple theory;[38] see Equa-
tions (1) and (2), where lc represents the critical hydro-
carbon chain length, n is the number of carbon atoms in
the hydrophobic tail, and R is the radius of the micelle. The
slightly larger diameters observed by DLS reflect the fact
that these values are a measure of the hydrodynamic dia-
meter of the micelle and not the unsolvated micelle. Both
theory and experiment illustrate that relatively small
changes in the length of the hydrophobic chain (2 carbons)
can result in observable differences in micelle size.
rescence intensity over a similar concentration range, con- lc Յ lmax ≈ (0.154 + 0.1265n) nm
(1)
sistent with lack of micelle formation. The CMCs measured
for these systems are quite low compared to other
R Յ lc
(2)
GdDOTA- and GdDTPA-based amphiphilic agents pre-
Given these measured diameters, the aggregation number
viously reported,[26–28,35] presumably due to the reduced po- (M) of each micelle can be estimated using Equation (3)
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Micelles with Enhanced CEST Sensitivity
1
High Resolution H-NMR Studies
High resolution 1H NMR spectroscopy (Figure 3) was
used to characterize the coordination geometries of the
Eu3+ complexes in these micelles. The macrocyclic axial
proton (H4 proton) resonance appearing near 26 ppm indi-
cates that both the monomeric and micellar complexes exist
largely as square antiprismatic (SAP) structures.[39] All pro-
1
ton resonances in the H-NMR spectra of Eu-(12), Eu-(14),
and Eu-(16) were broad in comparison to those observed
for Eu-(1), consistent with less motion in the higher molecu-
lar weight micellar species. Qualitatively, it was quite evi-
1
dent that all H NMR linewidths increased in parallel with
increasing alkyl carbon chain length. A resonance was not
observed for a Eu3+-bound water molecule in any of these
spectra but, given that the chemical shift of the Eu3+-shifted
water resonance is consistently found twofold further down-
field compared to the H4 proton resonance in all EuDOTA-
tetraamide complexes,[40] one would anticipate finding a
water exchange CEST peak near 52 ppm for each of these
complexes if water exchange is sufficiently slow.
Figure 2. DLS data showing the average diameters of micelles
formed from Eu-(12) (unfilled), Eu-(14) (vertical), and Eu-(16) (di-
agonal) measured at two different Eu3+ concentrations (5 and
10 mm), pH 7, 25 °C. The diameters estimated from basic theory
[eq. (2) and (3)] are shown in comparison.
and Equation (4), where ν corresponds to the hydrocarbon
chain volume. The aggregation number is particularly im-
portant since it provides an estimate of how many exchang-
ing groups are present per micelle.
CEST Studies
CEST spectra were collected on each complex (40 mm in
water) at different saturation powers (9.4, 14.1, 18.8, and
23.5 μT) and different temperatures. Given that the CMC
of all three amphiphilic compounds were in the 20–70 μm
range, it would have been impossible to investigate the
υ ≈ (27.4 + 26.9n)ϫ10–3 nm3
(3)
M = 4πR3/3υ
(4)
Using the theoretical diameters, the values of M for Eu- CEST properties of the monomeric forms of Eu-(12), Eu-
(12), Eu-(14) and Eu-(16) were calculated to be 56, 74, and (14), and Eu-(16) so it was assumed that the CEST proper-
94 molecules per micelle, respectively. This shows that PAR- ties of Eu-(1) would serve as an approximate model. Fig-
ACEST-based micelles can be prepared from 50–100 EuIII ure 4 shows the CEST spectra of Eu-(1), Eu-(12), Eu-(14),
complexes per micelle depending upon alkyl chain length, and Eu-(16) at 25 °C. It is obvious that all four compounds
a feature that may prove helpful in amplifying the CEST exhibit a strong water exchange peak near 53 ppm as antici-
signal from a single, targeted micelle.
pated from the 1H NMR studies. This observation suggests
Figure 3. High resolution 1H-NMR spectra of Eu-(1) (a), Eu-(12) (b), Eu-(14) (c), and Eu-(16) (d) recorded in D2O at 25 °C and 400 MHz.
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O. M. Evbuomwan, G. Kiefer, A. D. Sherry
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that changing the length of the hydrophobic chain had
minimal influence on the ligand field around the Eu3+ in
each of these complexes. One unanticipated but interesting
observation was that the linewidths of the Eu3+-bound
water exchange and the bulk water peaks are more narrow
in the spectrum of Eu-(16) than in the spectrum of Eu-(1)
or Eu-(12) or Eu-(14). This reflects a difference in the water
exchange kinetics in these complexes and suggests that
packing of the EuL headgroups in the amphiphilic micellar
systems does have an impact on the water exchange charac-
teristics of these agents.
Figure 5. CEST spectra of Eu-(1) (red squares), Eu-(12) (blue
circles), Eu-(14) (magenta upward triangles), and Eu-(16) (green
downward triangles) recorded at 37 °C, and pH 7.3. [Eu3+] =
40 mm, saturation power: 14.1 μT, and irradiation time: 6 s.
Figure 4. CEST spectra of 40 mm Eu-(1) (red squares), Eu-(12)
(blue circles), Eu-(14) (magenta upward triangles), and Eu-(16)
(green downward triangles) recorded at 25 °C, and pH 7.3. Satura-
tion power: 14.1 μT, and irradiation time: 6 s.
The water exchange CEST intensities of all four com-
pounds were similar at 25 °C but became well-differentiated
upon warming the samples to 37 °C. Here, the CEST spec-
tra registered pronounced differences both in intensity and
in shape (Figure 5). This behavior is typical of EuDOTA-
tetraamide complexes[9] and the differences observed here
demonstrate qualitatively that the water exchange rates in
these complexes falls in the order, Eu-(1) Ͼ Eu-(12) Ͼ Eu-
(14) Ͼ Eu-(16), with Eu-(16) being the slowest. The magni-
tude of CEST (Ms/Mo) at the optimal frequency at each
temperature is plotted vs. temperature in Figure 6. It is evi-
dent from these data that water exchange in Eu-(1) and Eu-
(12) do not become exchange limited at the lowest tempera-
ture examined here (25 °C) while water exchange in Eu-(14)
and Eu-(16) do appear to begin approaching an exchange
limit as the temperature nears 25 °C. These results demon-
strate that aggregation of a collection of Eu3+ chelates on
a micelle surface is beneficial for CEST by slowing water
exchange and thereby amplifying the CEST signal.
Figure 6. Plot of magnitude of CEST effect at 14.1 μT as a function
of temperature for Eu-(1) (squares), Eu-(12) (circles), Eu-(14) (up-
ward triangles), and Eu-(16) (downward triangles). [Eu3+] = 40 mm,
and irradiation time: 6 s.
change. This result was somewhat unexpected because pre-
vious studies have shown that increasing the bulkiness of
an amide substituent results in faster water exchange, pre-
sumably due to greater exposure of the coordination center
to bulk water.[34] Assuming a standard micelle model for
these systems, this suggests that EuL headgroups are inter-
A fit of these CEST spectra to the Bloch equations modi-
fied for chemical exchange provided the water residence life-
times (τm) summarized in Figure 7. From these data, the
activation energies of the water exchange process were de-
termined to be 69 kJ/mol, 82 kJ/mol, 86 kJ/mol, and 88 kJ/
mol for Eu-(1), Eu-(12), Eu-(14), and Eu-(16), respectively,
by plotting the natural log of kex vs. reciprocal temperature
(Supporting Information). These quantitative data support
the qualitative observations described above; water ex-
change slows down with an increase in alkyl chain length
Figure 7. Plot of water residence lifetimes (τm) as a function of
temperature for Eu-(1) (squares), Eu-(12) (circles), Eu-(14) (upward
triangles), and Eu-(16) (downward triangles). CEST spectra of
40 mm aqueous samples were recorded at pH 7, and 25 °C using a
saturation power of 14.1 μT and irradiation time of 6 s (see Sup-
porting Information). τm values were obtained by fitting CEST
as reflected in the higher activation energy for water ex- spectra to the Bloch equations modified for exchange.[31]
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Micelles with Enhanced CEST Sensitivity
acting with each other in a way that stabilizes the Eu3+
–
celle) obtained by fitting the CEST data to the Bloch equa-
water bond. The modest yet significant increase in bound tions were also found to increase with an increase in alkyl
water lifetime (τm) in micelles prepared from the C12 com- carbon chain length. Eu-(16) has the most promising CEST
plex vs. C16 complex again suggests that a higher density of features, the slowest water residence lifetime at 37 °C, the
EuL headgroups as indicated by differences in aggregation lowest CMC, and the highest micelle aggregation number.
numbers promotes slower water exchange.
The lowest detection limit for Eu-(16) was calculated to be
475 μm on a per Eu3+ basis and 5.3 μm on a per micelle
basis. Thus, micelle formation affords a 250-fold enhance-
ment in CEST sensitivity over a control compound having
a single methylamide substituent.
CEST Detection Limits
Plots of CEST intensity (Ms/Mo for the water exchange
peak) vs. Eu3+ concentration are shown for Eu-(1) and Eu-
(16) in Figure 8. These data were fit to an exponential func-
tion and the result was used to calculate the detection limit
Experimental Section
(DL) of each compound (defined as the concentration at General Remarks: All reagents and solvents were purchased from
commercial sources and used as received, unless otherwise stated.
1H and 13C NMR spectra were recorded on a Bruker AVANCE III
400 NMR spectrometer operating at 400.13 MHz and 100.03 MHz
respectively. Mass spectra were obtained by HT Laboratories San
Diego, California. Inductively coupled plasma measurements and
elemental analyses were performed by Galbraith Laboratories Inc.,
Knoxville, Tennessee. Fluorescence experiments were performed
using a Perkin–Elmer LS 50B Luminescence spectrometer at 25 °C.
Particle size analysis was performed at 25 °C using a Brookhaven
90Plus Particle Size Analyzer equipped with a 35 mm diode laser.
which a 5% CEST signal is observed). The DL of Eu-(1)
was estimated at 1300 μm while the DL of Eu-(16) was
about threefold lower, estimated at 475 μm. Given that the
latter concentration (475 μm) is well above the CMC of Eu-
(16), these differences can only be ascribed to the improved
water exchange characteristics of Eu-(16). Although it is yet
to be determined whether the micelles formed by Eu-(16)
are stable enough for in vivo use, given that there are ninety
Eu-(16) monomers per micelle, the detection limit of each
micelle is estimated at ca. 5.3 μm. This represents an ap- Melting points were determined using a Fisher–Johns melting point
apparatus.
proximate 250-fold sensitivity enhancement over Eu-(1).
General Procedure for the Synthesis of the N-Alkyl-2-bromoacet-
amides 3a–b, and 6a–d: A solution of bromoacetyl bromide
(1.2 equiv.) in dichloromethane (20 mL) was added dropwise to a
cooled solution (0 °C) of the corresponding amine (1 equiv.) and
K2CO3 (3 equiv.) in a mixture of dichloromethane (100 mL) and
water (50 mL). The resulting solution was warmed to room tem-
perature and stirred for 16 h after which the organic layer was
washed with water (2ϫ30 mL) and brine (1ϫ30 mL), dried with
Na2SO4, and concentrated in vacuo. The crude product was recrys-
tallized either from ethyl acetate or a mixture of methanol and
water to afford the corresponding bromoacetamide.
N-(2-Bromoacetyl)glycine Ethyl Ester (3a):[8] Colorless solid (13.1 g,
82%). 1H NMR (400 MHz, CDCl3, 25 °C): δ = 6.99 (br. s, 1 H,
3
3
NH), 4.17 (q, JH,H = 7 Hz, 2 H, OCH2CH3), 3.99 (d, JH,H
=
Figure 8. Plots of CEST signal as a function of Eu3+ concentration
for Eu-(1) (squares) and Eu-(16) (circles). The points were fit to
exponential functions (solid lines) and the resulting equations were
used to estimate the detection limit of each PARACEST agent.
3
5.2 Hz, 2 H, CH2NH), 3.85 (s, 2 H, CH2Br), 1.23 (t, JH,H = 7 Hz,
3 H, OCH2CH3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): δ =
14.1 (OCH2CH3), 28.5 (CH2Br), 41.9 (CH2NH), 61.7 (OCH2CH3),
165.8 (C=O), 169.2 (C=O) ppm. (ESMS ESI+): m/z (%) = 225
(100) [M + H]+.
N-(2-Bromoacetyl)glycine tert-Butyl Ester (3b): Colorless crystalline
Conclusion
solid (12.6 g, 84%); m.p. 90–94 °C. 1H NMR (400 MHz, CDCl3,
3
25 °C): δ = 7.00 (br. s, 1 H, NH), 3.96 (d, JH,H = 5.2 Hz, 2 H,
Four new EuDOTA-tetraamide complexes having a sin-
gle amide substituent with variable alkyl-carbon chain
lengths were synthesized and characterized by dynamic
light scattering, high resolution NMR and CEST spec-
troscopy. The three complexes having a single alkyl amide
side-chain with 12–16 carbons all form micelles at relatively
low concentrations. The CMCs determined with the aid of
NHCH2), 3.91 (s, 2 H, BrCH2), 1.48 [s, 9 H, C(CH3)3] ppm. 13C
NMR (100 MHz, CDCl3, 25 °C): δ = 28.0 [C(CH3)3], 28.7 (BrCH2),
42.6 (NHCH2), 82.8 [C(CH3)3], 165.6 (C=O), 168.3 (COO) ppm.
2-Bromo-N-methylacetamide (6a): Colorless crystalline solid (5.6 g,
49%); m.p. 41–45 °C. 1H NMR (400 MHz, CDCl3, 25 °C): δ = 6.81
3
(br. s, 1 H, NH), 3.84 (s, 2 H, BrCH2), 2.82 (d, JH,H = 4.9 Hz, 3
H, CH3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): δ = 26.9
a fluorescence probe showed that as the alkyl chain length (CH3), 29.0 (BrCH2), 166.3 (C=O) ppm. (ESMS ESI+): m/z (%) =
152 (100) [M + H]+.
was increased, the CMC decreased as expected. DLS data
confirmed the formation of micelles in solution, the size of
which increased with an increase in alkyl carbon chain
length. The water residence lifetimes in each complex (mi- δ = 6.50 (br. s, 1 H, NH), 3.89 (s, 2 H, BrCH2), 3.30 (q, JH,H
2-Bromo-N-dodecylacetamide (6b): Colorless, crystalline solid
1
(7.0 g, 85%); m.p. 51–55 °C. H NMR (400 MHz, CDCl3, 25 °C):
3
=
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O. M. Evbuomwan, G. Kiefer, A. D. Sherry
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7 Hz, 2 H, NHCH2), 1.56 (m, 2 H, NH2CH2CH2), 1.27 [br. s, 18
Ph), 128.2 (3-Ph), 128.6 (2-Ph), 136.5 (1-Ph), 156.5 (NCOO), 169.1
(C=O), 171.1 (CH2COO) ppm. (ESMS ESI+): m/z (%) = 821 (100)
3
H, (CH2)9CH3], 0.88 (t, JH,H = 7 Hz, 3 H, CH2CH3) ppm. 13C
NMR (100 MHz, CDCl3, 25 °C): δ = 14.1 (CH3), 22.7 (CH2CH3), [M + H]+.
26.8–29.6 [(CH2)9CH2CH3], 31.9 (BrCH2), 40.3 (NHCH2), 165.1
Acetamide 5b: A procedure analogous to that of compound 5a was
(C=O) ppm. (ESMS ESI+): m/z (%) = 308 (100) [M + 2H]+.
followed. A tan hygroscopic solid was obtained (quantitative yield).
1H NMR (400 MHz, CDCl3, 25 °C): δ = 9.10 (br. s, 1 H, NH),
8.23 (br. s, 2 H, NH), 3.82 (br. m, 6 H, CH2COO), 3.72 (br. m, 6
H, NCH2C=O), 3.04–3.37 (br. s, 16 H, ring CH2N), 1.37 [s, 27 H,
C(CH3)3] ppm. 13C NMR (100 MHz, CDCl3, 25 °C): δ = 28.1
[C(CH3)3], 41.9 (CH2COO), 49.3–52.6 (ring CH2N), 57.0
(CH2C=O), 81.9 [C(CH3)3], 169.4 (C=O), 171.5 (CH2COO) ppm.
(ESMS ESI+): m/z (%) = 686 (100) [M + H]+.
2-Bromo-N-tetradecylacetamide (6c): Colorless solid (7.2 g, 86%);
1
m.p. 48–50 °C. H NMR (400 MHz, CDCl3, 25 °C): δ = 6.40 (br.
3
s, 1 H, NH), 3.81 (s, 2 H, BrCH2), 3.21 (q, JH,H = 7 Hz, 2 H,
NHCH2), 1.47 (m, 2 H, NHCH2CH2), 1.19 [s, 22 H, (CH2)9CH3],
3
0.81 (t, JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz,
CDCl3, 25 °C): δ = 14.1 (CH3), 22.7 (CH2CH3), 26.8 [NH(CH2)2-
CH2], 29.3–29.7 [(CH2)9CH2CH3], 31.9 (NHCH2CH2), 38.1
(BrCH2), 40.3 (NHCH2), 165.1 (C=O) ppm.
Acetamide 7a: Compound 5a (2.3 g, 3.84 mmol) was added to a
mixture of compound 6a (0.59 g, 3.84 mmol) and K2CO3 (1.1 g,
7.7 mmol) in anhydrous acetonitrile (90 mL) and the reaction was
heated to reflux (60 °C) for 6 h. The solvent was removed in vacuo
and the resulting residue was taken up in dichloromethane and
washed with water (2ϫ30 mL) and brine (1ϫ30 mL). The dichlo-
romethane layer was dried with Na2SO4, the solvent was removed
in vacuo to yield the titled compound as a brown hygroscopic solid
(1.25 g, 48%) which was used without further purification. 1H
NMR (400 MHz, CDCl3, 25 °C): δ = 7.60, 7.53, and 6.99 (br. s, 4
2-Bromo-N-hexadecylacetamide (6d): Colorless solid (7.4 g, 99%);
1
m.p. 61–65 °C. H NMR (400 MHz, CDCl3, 25 °C): δ = 6.42 (br.
3
s, 1 H, NH), 3.81 (s, 2 H, BrCH2), 3.20 (q, JH,H = 7 Hz, 2 H,
NHCH2), 1.46 (m, 2 H, NHCH2CH2), 1.19 [s, 26 H, (CH2)9CH3],
3
0.81 (t, JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz,
CDCl3, 25 °C): δ = 14.1 (CH3), 22.7 (CH2CH3), 27.01 [NH(CH2)2-
CH2], 29.2–29.7 [(CH2)11CH2CH3], 31.9 (NHCH2CH2), 39.3
(BrCH2), 40.3 (NHCH2), 165.1 (C=O) ppm.
Carbamate 4a: To a solution of compound 2 (4.3 g, 13.7 mmol)
and K2CO3 (7.6 g, 54.9 mmol) in anhydrous acetonitrile (250 mL)
was added compound 3a (9.2 g, 41.1 mmol). The reaction mixture
was heated to reflux (65 °C) for 18 h and the solvents were removed
in vacuo. The residue was dissolved in dichloromethane (100 mL)
and washed with water (2ϫ50 mL). The organic layer was dried
with Na2SO4, the solvents were removed in vacuo to yield a brown
hygroscopic solid (9.3 g, 92%) which was used without further puri-
3
3
H, NH), 4.10 (q, JH,H = 7 Hz, 6 H, CH2CH3), 3.93 (d, JH,H
=
5.2 Hz, 6 H, CH2COO), 3.03–3.07 (m, 8 H, NCH2C=O), 2.65 (br.
3
s, 3 H, CH3), 2.62 (br. s, 16 H, ring CH2N), 1.21 (t, JH,H = 7 Hz,
9 H, CH2CH3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C): δ = 14.1
(CH2CH3), 25.8 (CH3), 40.7 (CH2COO), 53.1–53.4 (ring CH2N),
58.9, and 59.1 (NCH2C=O), 61.4 (CH2CH3), 170.1 (C=ONH),
171.5 (COO) ppm. (ESMS ESI+): m/z (%) = 674 (100) [M + H]+,
696 (20) [M + Na]+. C29H52N8O10·KCl (746.31): calcd. C 46.6, H
7.0, N 14.9; found C 46.3, H 6.7, N 14.7%.
1
fication. H NMR (400 MHz, CDCl3, 25 °C): δ = 7.42 (br. s, 3 H,
3
NH), 7.26 (m, 5 H, Ph), 5.0 (s, 2 H, OCH2Ph), 4.09 (q, JH,H
=
3
7 Hz, 6 H, CH2CH3), 3.87 and 3.91 (br. d, JH,H = 5.2 Hz, 6 H,
CH2COO), 3.51 (br. s, 6 H, NCH2C=O), 2.67–3.16 (br. s, 16 H,
Acetamide 7b: A procedure analogous to that for compound 7a
was followed. The residue was precipitated from ethyl acetate to
afford the title compound as a tan solid (2.0 g, 93%); m.p. 121–
ring CH2N), 1.19 (t, JH,H = 7 Hz, 9 H, CH3) ppm. 13C NMR
3
(100 MHz, CDCl3, 25 °C): δ = 14.1 (CH3), 40.8 (CH2COO), 47.3–
53.9 (ring CH2N), 58.8 (CH2C=O), 61.4 (CH2CH3), 67.4
(OCH2Ph), 128.1 (4-Ph), 128.2 (3-Ph), 128.6 (2-Ph), 136.4 (1-Ph),
156.5 (NCOO), 170.0 (C=O), 171.0 (CH2COO) ppm. (ESMS
ESI+): m/z (%) = 737 (100) [M + H]+, 759 (28) [M + Na]+.
1
124 °C. H NMR (400 MHz, CDCl3, 25 °C): δ = 8.30–7.07 (br. s,
3
4 H, NH), 4.04 (q, JH,H = 7 Hz, 6 H, CH2CH3), 3.89 (br. s, 6 H,
CH2COO), 3.21 (m, 2 H, NHCH2CH2) 3.09 (s, 8 H, NCH2C=O),
2.43–2.65 (br. s, 16 H, ring CH2N), 1.39 (m, 2 H, NHCH2CH2),
3
1.19 [br. s, 18 H, (CH2)9CH3] 1.17 (t, JH,H = 7 Hz, 9 H,
3
OCH2CH3), 0.81 (t, JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR
Acetamide 5a: Compound 4a (5.7 g, 7.8 mmol) was dissolved in
ethanol (75 mL), and 10% palladium on carbon (0.6 g) was added.
The mixture was shaken on a Parr hydrogenator under a hydrogen
pressure of 55 psi at room temperature for 48 h. The reaction was
filtered, and the solvents were removed in vacuo to afford the title
(100 MHz, CDCl3, 25 °C): δ = 14.1 (CH3), 22.6 (CH2CH3), 27.1
[NH(CH2)2CH2], 29.3–29.6 [(CH2)7CH3], 31.9 (NHCH2CH2), 39.6
(NHCH2CH2), 41.2 (CH2COO), 50.6 (ring CH2N), 57.3 and 57.6
(NCH2C=O), 61.3 (OCH2CH3), 169.9 [C=ONH(CH2)11CH3],
171.1 (C=ONHCH2COO) 172.2 (CH2COO) ppm. (ESMS ESI+):
m/z (%) = 850 (100) [M + Na]+. C40H74N8O10·H2O·NaCl (902.52):
calcd. C 53.2, H 8.5, N 12.4; found C 53.4, H 8.3, N 12.1%.
1
compound as a tan oil (4.6 g, 99%). H NMR (400 MHz, CDCl3,
3
25 °C): δ = 7.71 (br. s, 3 H, NH), 4.10 (q, JH,H = 7 Hz, 6 H,
3
CH2CH3), 3.84 and 3.92 (br. d, JH,H = 5.2 Hz, 6 H, CH2COO),
3.17 and 3.20 (s, 6 H, NCH2C=O), 2.60–2.80 (br. s, 16 H, ring
Acetamide 7c: A procedure analogous to that for compound 7a was
followed. The residue was purified by column chromatography over
silica gel Rf = 0.4 (CH2Cl2/MeOH, 100:10) to afford the title com-
pound as a tan solid (2.2 g, 63%); m.p. 142–144 °C. 1H NMR
3
CH2N), 1.20 (t, JH,H = 7 Hz, 9 H, CH3) ppm. 13C NMR
(100 MHz, CDCl3, 25 °C): δ = 14.1 (CH3), 40.9 (CH2COO), 51.7–
57.4 (ring CH2N), 58.3 and 59.8 (NCH2C=O), 61.4 (CH2CH3),
170.1 and 170.2 (C=O), 171.6 and 171.9 (CH2COO) ppm. (ESMS
ESI+): m/z (%) = 601 (100) [M + H]+.
3
(400 MHz, MeOD, 25 °C): δ = 4.16 (q, JH,H = 7 Hz, 6 H,
CH2CH3), 3.93 (br. s, 6 H, CH2COO), 3.20 (m, 2 H, NHCH2CH2),
3.15 (s, 8 H, NCH2C=O), 2.43–2.70 (br. s, 16 H, ring CH2N), 1.48
(m, 2 H, NHCH2CH2), 1.29 [br. s, 22 H, (CH2)11CH3] 1.26 (t, 3JH,H
Carbamate 4b: A procedure analogous to that from compound 4a
was followed. An off-white solid was ontained (90%). 1H NMR
3
3
(400 MHz, CDCl3, 25 °C): δ = 7.52 (br. t, JH,H = 5.6 Hz, 1 H,
= 7 Hz, 9 H, OCH2CH3), 0.90 (t, JH,H = 7 Hz, 3 H, CH3) ppm.
3
NH), 7.37 (m, 5 H, Ph), 7.34 (br. t, JH,H = 5.6 Hz, 2 H, NH), 5.1
13C NMR (100 MHz, MeOD, 25 °C): δ = 14.4 and 14.6 (CH3), 23.7
(CH2CH3), 28.1 [NH(CH2)2CH2], 30.4–30.8 [(CH2)9CH3], 33.1
3
(s, 2 H, OCH2Ph), 3.87 and 3.92 (br. d, JH,H = 5.6 Hz, 6 H,
CH2COO), 3.56 (br. s, 6 H, NCH2C=O), 2.70–3.23 (br. s, 16 H, (NHCH2CH2), 40.4 (NHCH2CH2), 42.0 (CH2COO), 51.7 (ring
ring CH2N), 1.46 [s, 27 H, C(CH3)3] ppm. 13C NMR (100 MHz, CH2N), 58.2 and 58.3 (NCH2C=O), 62.3 (OCH2CH3), 171.5
CDCl3, 25 °C): δ = 28.1 [C(CH3)3], 41.7 (CH2COO), 53.9 (ring
[C=ONH(CH2)13CH3],
173.3
(C=ONHCH2COO)
174.3
CH2N), 58.9 (CH2C=O), 67.3 (OCH2Ph), 82.1 [C(CH3)3], 128.1 (4- (CH2COO) ppm. (ESMS ESI+): m/z (%) = 878 (100) [M + Na]+.
2132
www.eurjic.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2012, 2126–2134

Micelles with Enhanced CEST Sensitivity
C42H78N8O10·H2O·CH2Cl2 (956.55): calcd. C 53.9, H 8.6, N 11.7;
found C 53.8, H 8.4, N 11.6%.
31.9 (NHCH2CH2), 39.1 (NHCH2CH2), 43.5 (CH2COO), 51.2
(ring CH2N), 57.4 (NCH2C=O), 171.2 [C=ONH(CH2)15CH3],
173.1 (C=ONHCH2COO) 176.1 (COO) ppm. (ESMS ESI-): m/z
(%) = 797 (100) [M – H]–. Elemental analysis was not performed
due to high salt content.
Acetamide 7d: A procedure analogous to that for compound 7a
was followed. A tan hygroscopic solid was obtained. 1H NMR
(400 MHz, CDCl3, 25 °C): δ = 7.60 (br. s, 2 H, NH), 7.53 (br. s, 1
H, NH), 6.93 (br. s, 1 H, NH), 3.88 (br. s, 6 H, CH2COO), 3.19
(m, 2 H, NHCH2CH2), 3.12 (m, 6 H, NCH2C=O), 2.73 (br. s, 16
H, ring CH2N), 1.45 [s, 27 H, C(CH3)3], 1.24 [br. s, 26 H, (CH2)13-
CH3], 0.87 (t, 3JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz,
CDCl3, 25 °C): δ = 14.1 (CH3), 22.7 (CH2CH3), 27.1 [NH(CH2)2-
CH2], 28.1 [C(CH3)3], 29.3–29.8 [(CH2)11CH3], 31.9 (NHCH2CH2),
39.2 (NHCH2CH2), 41.7 (CH2COO), 53.2 (ring CH2N), 59.3
(CH2C=O), 81.9 [C(CH3)3], 169.2 (C=O), 171.3 (CH2COO) ppm.
(ESMS ESI+): m/z (%) = 967 (98) [M + H]+, 989 (100) [M +
Na]+. C50H94N8O10·0.5CH3OH (982.73): calcd. C 61.7, H 9.8, N
11.4; found C 61.9, H 9.2, N 11.0%.
General Procedure for the Formation of the Europium(III) Com-
plexes: To a solution of EuCl3 (0.9 equiv.) in water, was added the
corresponding ligand (1 equiv.) and the mixture was stirred at 50 °C
for 18 h while the pH was maintained at 5.5–6. The absence of
excess metal was confirmed with the aid of a xylenol orange test
after which the solution was lyophilized to yield the respective com-
plexes as tan solids.
Eu-(1): 1H NMR (400 MHz, D2O, 25 °C): δ = 26.4 (br. d), –2.52
(br. t), –4.85 (br. d), –5.9 (br. d), –8.25 (br. t), –10.9 (br. s), –13.3
(br. t) ppm. (ESMS ESI+): m/z (%) = 739 (100) [M – 2H]+. Free
ligand was present at less than 20 as estimated by NMR spec-
troscopy.
Acetamide 1: Compound 7a (1.2 g, 1.82 mmol) was added to a
LiOH solution (0.2 m, 36 mL) at 0 °C. The solution was warmed
up to room temperature and stirred for 16 h. The resulting sample
was lyophilized to afford the title compound in quantitative yield.
1H NMR (400 MHz, D2O, 25 °C): δ = 3.64 and 3.59 (br. s, 6 H,
CH2COO), 2.99–3.09 (br. s, 8 H, NCH2C=O) 2.39 and 2.59 (br. s,
16 H, ring CH2N), 2.55 (s, 3 H, CH3) ppm. 13C NMR (100 MHz,
D2O, 25 °C): δ = 25.7 (CH3), 43.2 (CH2COO), 50.4 (ring CH2N),
56.2–57.7 (NCH2C=O), 173.3 (C=ONHCH3), 173.9 (C=ONH-
CH2COO) 176.8 (COO) ppm. (ESMS ESI+): m/z (%) = 589 (100)
[M + H]+. Elemental analysis was not performed due to high salt
content.
1
Eu-(12): H NMR (400 MHz, D2O, 25 °C): δ = 25.7 (br. d), –2.35
(br. t), –4.35 (br. s), –5.85 (br. d), –8.33 (br. d), –10.5 (br. d), –12.5
(br. d), –13.1 (br. d). (ESMS ESI+): m/z (%) = 893 (100) [M –
2H]+. Free ligand was present at less than 20 as estimated by NMR
spectroscopy.
1
Eu-(14): H NMR (400 MHz, D2O, 25 °C): δ = 25.7 (br. s), –2.30
(br. t), –4.11 (br. s), 5.52 (br. d), –7.9 (br. m), –10.1 (br. m), –12.5
(br. d) ppm. (ESMS ESI+): m/z (%) = 921 (100) [M – 2H]+. Free
ligand was present at less than 20 as estimated by NMR spec-
troscopy.
Acetamide 12: A procedure analogous to that for compound 1 was
followed using compound 7b. H NMR (400 MHz, D2O, 25 °C): δ
1
Eu-(16): H NMR (400 MHz, D2O, 25 °C): δ = 25.7 (br. s), –2.51
1
(br. t), –4.04 (br. m), –8.23 (br. d), –10.5 (br. t), –12.8 (br. d).
(ESMS ESI+): m/z (%) = 949 (100) [M – 2H]+. Free ligand was
present at less than 20 as estimated by NMR spectroscopy.
= 3.73 and 3.69 (br. s, 6 H, CH2COO), 3.22 (s, 8 H, NCH2C=O)
3.10 (m, 2 H, NHCH2CH2), 2.54–2.70 (br. s, 16 H, ring CH2N),
1.43 (m, 2 H, NHCH2CH2), 1.22 [br. s, 18 H, (CH2)9CH3], 0.81 (t,
3JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz, D2O, 25 °C):
δ = 13.9 (CH3), 22.5 (CH2CH3), 26.8 [NH(CH2)2CH2], 29.0–29.6
[(CH2)7CH3], 31.8 (NHCH2CH2), 39.3 (NHCH2CH2), 43.2 and
43.5 (CH2COO), 50.3 (ring CH2N), 57.0 and 56.6 (NCH2C=O),
171.8 [C=ONH(CH2)11CH3], 173.4 (C=ONHCH2COO) 176.1
(COO) ppm. (ESMS, ESI-): m/z (%) = 741 (100) [M – H]–. Elemen-
tal analysis was not performed due to high salt content.
Fluorescence Studies: Critical micelle concentrations (CMC) were
determined by fluorescence spectroscopy using a well established
procedure.[30] Emission spectra were recorded at room temperature
using a 1.0 cm quartz cell, an excitation wavelength of 350 nm, scan
rate of 200 nm/min, and excitation and emission slit widths of 10
and 5 nm, respectively. 8-Anilino-1-naphthalenesulfonic acid
(ANS) was used as the fluorescent probe. Aqueous stock solutions
of ANS and the europium complexes were prepared using borate
buffer (pH 8.5). Fluorescence titrations were performed by adding
small aliquots of the amphiphile stock solution to 1 mL of 20 μm
ANS with the appropriate corrections made for dilution. The CMC
values were determined by plotting the relative fluorescence inten-
sity of ANS at 480 nm, upon excitation at 350 nm as a function of
europium(III) concentration.
Acetamide 14: A procedure analogous to that for compound 1 was
1
followed using compound 7c. H NMR (400 MHz, D2O, 25 °C): δ
= 3.71 and 3.64 (br. s, 6 H, CH2COO), 3.21 (s, 8 H, NCH2C=O)
3.12 (m, 2 H, NHCH2CH2), 2.58 (br. s, 16 H, ring CH2N), 1.40
(m, 2 H, NHCH2CH2), 1.19 [br. s, 22 H, (CH2)11CH3], 0.80 (t,
3JH,H = 7 Hz, 3 H, CH3) ppm. 13C NMR (100 MHz, D2O, 25 °C):
δ = 14.0 (CH3), 22.6 (CH2CH3), 26.9 [NH(CH2)2CH2], 29.1–29.8
[(CH2)9CH3], 31.9 (NHCH2CH2), 39.1 (NHCH2CH2), 43.1 and
43.4 (CH2COO), 51.1 (ring CH2N), 57.3 and 56.3 (NCH2C=O),
171.4 [C=ONH(CH2)13CH3], 173.1 (C=ONHCH2COO) 176.1
(COO) ppm. (ESMS, ESI-): m/z (%) = 769 (100) [M – H]–. Elemen-
tal analysis was not performed due to high salt content.
Dynamic Light Scattering (DLS): DLS was used to characterize
micelle size. Samples were prepared by dissolving the appropriate
amount of each agent in DI water to yield Eu3+ concentrations
of 5 mm and 10 mm. 200 μL aliquots were transferred into plastic
cuvettes with 1 cm path lengths, and analyzed at a pH of 7.3 (MES
buffer) and 25 °C.
Acetamide 16: Compound 7d was suspended in TFA and stirred
for 24 h at room temperature. The TFA was removed in vacuo and
the residue was washed with diethyl ether, and dissolved in water.
The resulting sample was lyophilized to afford the title compound
in quantitative yield. 1H NMR (400 MHz, D2O, 25 °C): δ = 3.71
CEST Experiments: CEST spectra were generated by applying a
frequency-selective saturation pulse in 1 ppm increments over a
200 ppm frequency range, and plotting the residual bulk water sig-
nal intensity (Ms/Mo) as a function of saturation frequency. CEST
and 3.64 (br. s, 6 H, CH2COO), 3.21 (s, 8 H, NCH2C=O) 3.12 (m, studies were carried out at 25 °C and pH 7.4 (MES buffer) using
2 H, NHCH2CH2), 2.58 (br. s, 16 H, ring CH2N), 1.40 (m, 2 H,
four different B1 power levels (9.4, 14.1, 18.8, and 23.5 μT), and a
NHCH2CH2), 1.19 [br. s, 26 H, (CH2)13CH3] 0.80 (t, 3JH,H = 7 Hz, presaturation time of 6 s. The CEST properties of each sample were
3 H, CH3) ppm. 13C NMR (100 MHz, D2O, 25 °C): δ = 14.0 (CH3), also investigated as a function of temperature using a B1 of 14.1
22.7 (CH2CH3), 26.9 [NH(CH2)2CH2], 29.2–29.9 [(CH2)11CH3], μT, pH 7.4, and a 6 s presaturation time.
Eur. J. Inorg. Chem. 2012, 2126–2134
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
2133

O. M. Evbuomwan, G. Kiefer, A. D. Sherry
FULL PAPER
[17]
J. A. Pikkemaat, R. T. Wegh, R. Lamerichs, R. A. van de Mo-
lengraaf, S. Langereis, D. Burdinski, A. Y. F. Raymond, H. M.
Janssen, B. F. M. de Waal, N. P. Willard, E. W. Meijer, H.
Grull, Contrast Media Mol. Imaging 2007, 2, 229.
K. Snoussi, J. W. M. Bulte, M. Gueron, P. C. M. van Zijl,
Magn. Reson. Med. 2003, 49, 998.
Y. K. Wu, P. Y. Zhao, G. E. Kiefer, A. D. Sherry, Macromole-
cules 2010, 43, 6616.
Y. K. Wu, Y. F. Zhou, O. Ouari, M. Woods, P. Y. Zhao, T. C.
Soesbe, G. E. Kiefer, A. D. Sherry, J. Am. Chem. Soc. 2008,
130, 13854.
S. Aime, D. D. Castelli, E. Terreno, Angew. Chem. 2005, 117,
5649; Angew. Chem. Int. Ed. 2005, 44, 5513.
O. Vasalatiy, R. D. Gerard, P. Zhao, X. K. Sun, A. D. Sherry,
Bioconjugate Chem. 2008, 19, 598.
O. Vasalatiy, P. Zhao, S. Zhang, S. Aime, A. D. Sherry, Contrast
Media Mol. Imaging 2006, 1, 10.
P. M. Winter, K. J. Cai, J. Chen, C. R. Adair, G. E. Kiefer, P. S.
Athey, P. J. Gaffney, C. E. Buff, J. D. Robertson, S. D. Caru-
thers, S. A. Wickline, G. M. Lanza, Magn. Reson. Med. 2006,
56, 1384.
A. Accardo, D. Tesauro, L. Aloj, C. Pedone, G. Morelli, Coord.
Chem. Rev. 2009, 253, 2193.
J. P. Andre, E. Toth, H. Fischer, A. Seelig, H. R. Macke, A. E.
Merbach, Chem. Eur. J. 1999, 5, 2977.
R. Hovland, C. Glogard, A. J. Aasen, J. Klaveness, Org. Bi-
omol. Chem. 2003, 1, 644.
G. M. Nicolle, E. Toth, K. P. Eisenwiener, H. R. Macke, A. E.
Merbach, J. Biol. Inorg. Chem. 2002, 7, 757.
Fitting CEST Spectra to Obtain Bound Water Lifetimes: The water
residence lifetime (τm) of each complex was evaluated by fitting the
CEST spectra collected at multiple B1’s to a three pool model
(Eu3+-bound water, ligand amide protons, and bulk water) based
on the numerical solutions to the Bloch equations.[31] The T1 and
T2 of bulk water (determined experimentally), the bound water and
amide proton concentrations, presaturation time (in seconds) and
saturation power (in Hz) were all included in the fitting procedure.
[18]
[19]
[20]
Supporting Information (see footnote on the first page of this arti-
cle): Plots of fluorescence intensity vs. Eu3+ concentration, calcula-
tion of CMCs, full CEST spectra recorded at different saturation
powers and different temperatures, detection limit plots and calcu-
lations, plot of natural log of kex vs. reciprocal temperature and
calculation of the activation energies for Eu-(1), Eu-(12), Eu-(14),
and Eu-(16).
[21]
[22]
[23]
[24]
Acknowledgments
This work was supported in part by grants from the National Insti-
tutes of Health (NIH) (CA-115531, RR-02584, and EB-00482) and
the Robert A. Welch Foundation (AT-584).
[25]
[26]
[27]
[28]
[1] A. E. Merbach, É. Tóth, The chemistry of contrast agents in
medical magnetic resonance imaging, Wiley, Chichester, UK,
2001.
[2] A. D. Sherry, M. Woods, Annu. Rev. Biomed. Eng. 2008, 10,
12.1.
[3] V. Guivel-Scharen, T. Sinnwell, S. D. Wolff, R. S. Balaban, J.
Magn. Reson. 1998, 133, 36.
[4] K. M. Ward, A. H. Aletras, R. S. Balaban, J. Magn. Reson.
2000, 143, 79.
[5] S. Zhang, K. Wu, M. C. Biewer, A. D. Sherry, Inorg. Chem.
2001, 40, 4284.
[6] S. R. Zhang, M. Merritt, D. E. Woessner, R. E. Lenkinski,
A. D. Sherry, Acc. Chem. Res. 2003, 36, 783.
[7] S. Zhang, P. Winter, K. Wu, A. D. Sherry, J. Am. Chem. Soc.
2001, 123, 1517.
[8] S. Aime, A. Barge, D. D. Castelli, F. Fedeli, A. Mortillaro, F. U.
Nielsen, E. Terreno, Magn. Reson. Med. 2002, 47, 639.
[9] S. R. Zhang, C. R. Malloy, A. D. Sherry, J. Am. Chem. Soc.
2005, 127, 17572.
[10] R. Trokowski, J. M. Ren, F. K. Kalman, A. D. Sherry, Angew.
Chem. 2005, 117, 7080; Angew. Chem. Int. Ed. 2005, 44, 6920.
[11] S. Aime, D. Delli Castelli, F. Fedeli, E. Terreno, J. Am. Chem.
Soc. 2002, 124, 9364.
[29] F. Kielar, L. Tei, E. Terreno, M. Botta, J. Am. Chem. Soc. 2010,
132, 7836.
[30] E. Devendittis, G. Palumbo, G. Parlato, V. Bocchini, Anal. Bio-
chem. 1981, 115, 278.
[31] D. E. Woessner, S. R. Zhang, M. E. Merritt, A. D. Sherry,
Magn. Reson. Med. 2005, 53, 790.
[32] M. Woods, G. E. Kiefer, S. Bott, A. Castillo-Muzquiz, C. Esh-
elbrenner, L. Michaudet, K. McMillan, S. D. K. Mudigunda,
D. Grin, G. Tircso, S. R. Zhang, P. Zhao, A. D. Sherry, J. Am.
Chem. Soc. 2004, 126, 9248.
[33] S. Aime, A. Barge, A. S. Batsanov, M. Botta, D. D. Castelli, F.
Fedeli, A. Mortillaro, D. Parker, H. Puschmann, Chem. Com-
mun. 2002, 1120.
[34] T. Mani, G. Tircso, O. Togao, P. Zhao, T. C. Soesbe, M. Takah-
ashi, A. D. Sherry, Contrast Media Mol. Imaging 2009, 4, 183.
[35] C. Glogard, R. Hovland, S. L. Fossheim, A. J. Aasen, J. Klave-
ness, J. Chem. Soc. Perkin Trans. 2 2000, 5, 1047.
[36] T. Geva, G. F. Greil, A. C. Marshall, M. Landzberg, A. J. Pow-
ell, Circulation 2002, 106, 473.
[12] R. Trokowski, S. R. Zhang, A. D. Sherry, Bioconjugate Chem.
2004, 15, 1431.
[13] T. Chauvin, P. Durand, M. Bernier, H. Meudal, B. T. Doan, F.
Noury, B. Badet, J. C. Beloeil, E. Toth, Angew. Chem. 2008,
120, 4442; Angew. Chem. Int. Ed. 2008, 47, 4370.
[14] Y. Li, V. R. Sheth, G. Liu, M. D. Pagel, Contrast Media Mol.
Imaging 2011, 6, 219.
[15] B. Yoo, M. D. Pagel, J. Am. Chem. Soc. 2006, 128, 14032.
[16] S. Aime, D. Delli Castelli, E. Terreno, Angew. Chem. 2003, 115,
4665; Angew. Chem. Int. Ed. 2003, 42, 4527.
[37] M. Vaccaro, A. Accardo, D. Tesauro, G. Mangiapia, D. Lof,
K. Schillen, O. Soderman, G. Morelli, L. Paduano, Langmuir
2006, 22, 6635.
[38] J. N. Israelachvili, Intermolecular and surface forces, 3rd ed.,
Academic Press, Burlington, MA.
[39] S. Aime, A. Barge, M. Botta, A. S. De Sousa, D. Parker, Angew.
Chem. 1998, 110, 2819; Angew. Chem. Int. Ed. 1998, 37, 2673.
[40] S. R. Zhang, A. D. Sherry, J. Solid State Chem. 2003, 171, 38.
Received: December 8, 2011
Published Online: February 9, 2012
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