Mechanisms of ZnII-activated magnetic resonance imaging agents

Article abstract of DOI:10.1021/ic801458u

We report on the mechanism of a series of Zn^II-activated magnetic resonance contrast agents that modulate the access of water to a paramagnetic Gd^III ion to create an increase in relaxivity upon binding of Zn^II. In the absence and presence of Zn^II, the coordination at the Gd^III center is modulated by appended Zn ^II binding groups. These groups were systematically varied to optimize the change in coordination upon Zn^II binding. We observe that at least one appended aminoacetate must be present as a coordinating group to bind Gd^III and effectively inhibit access of water. At least two binding groups are required to efficiently bind Zn^II, creating an unsaturated complex and allowing access of water. ¹³C isotopic labeling of the acetate binding groups for NMR spectroscopy provides evidence of a change in the metal coordination of these groups upon the addition of Zn ^II supporting our proposed mechanism of activation as presented.

Full text of DOI:10.1021/ic801458u

Inorg. Chem. 2008, 47, 10788-10795
Mechanisms of ZnII-Activated Magnetic Resonance Imaging Agents
Jody L. Major, Rene M. Boiteau, and Thomas J. Meade*
Department of Chemistry, Biochemistry and Molecular Cell Biology, Neurobiology and
Physiology, and Radiology, Northwestern UniVersity, 2145 Sheridan Road,
EVanston, Illinois 60208-3113
Received August 1, 2008
We report on the mechanism of a series of ZnII-activated magnetic resonance contrast agents that modulate the
access of water to a paramagnetic GdIII ion to create an increase in relaxivity upon binding of ZnII. In the absence
and presence of ZnII, the coordination at the GdIII center is modulated by appended ZnII binding groups. These
groups were systematically varied to optimize the change in coordination upon ZnII binding. We observe that at
least one appended aminoacetate must be present as a coordinating group to bind GdIII and effectively inhibit
access of water. At least two binding groups are required to efficiently bind ZnII, creating an unsaturated complex
and allowing access of water. 13C isotopic labeling of the acetate binding groups for NMR spectroscopy provides
evidence of a change in the metal coordination of these groups upon the addition of ZnII supporting our proposed
mechanism of activation as presented.
Introduction
to image biological processes in vivo, our laboratory
pioneered the development of a series of activated contrast
agents sensitive to enzymatic activity and secondary mes-
sengers.5-10 These activated contrast agents exploit the
modulation of coordinated water molecules to produce
distinct relaxation states in response to a physiological event.
Ideally, a q-modulated MRI contrast agent has two distinct
states, a low relaxivity state (q ) 0) before activation and a
high relaxivity state (q ) 1 or 2) after activation, to produce
an increase in the observed MRI signal intensity.
ZnII plays a critical role in cellular physiology and is
involved in structural stability, catalytic activity, and signal
transduction processes.11-13 The release of high concentra-
tions of ZnII (200-300 µM) from neuronal synaptic vessels
Magnetic resonance imaging (MRI) is a noninvasive
technique capable of producing three-dimensional images
of opaque organisms with excellent spatial and temporal
1
1
resolution. MRI measures the H NMR signal of water,
where the signal intensity is proportional to the relaxation
rates of the nuclear spins. Variation in the water concentration
and local environment contributes to the vivid contrast
observed in an acquired image. Intrinsic contrast can be
augmented by employing paramagnetic contrast agents that
are designed to accelerate the relaxation of water protons.1,2
Typically, GdIII chelates are used as a result of having seven
unpaired electrons and a symmetrical S ground state. The
efficiency of these complexes to decrease the T1 of water
protons is reported by the relaxivity value, r1 (mM-1 s-1).1,2
The Solomon-Bloembergen-Morgan theory describes
several variables that can be manipulated to produce changes
in the relaxivity of a GdIII chelate and include the hydration
number (q), the mean residence lifetime of coordinated
waters (τm), and the rotational correlation time (τr).3,4 In order
(4) Bloembergen, N.; Morgan, L. O. J. Chem. Phys. 1961, 34, 842–850.
(5) Li, W.-H.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc. 1999, 121,
1413–1414.
(6) Li, W.-H.; Parigi, G.; Fragai, M.; Luchinat, C.; Meade, T. J. Inorg.
Chem. 2002, 41, 4018–4024.
(7) Louie, A. Y.; Huber, M. M.; Ahrens, E. T.; Rothbacher, U.; Moats,
R.; Jacobs, R. E.; Fraser, S. E.; Meade, T. J. Nat. Biotechnol. 2000,
18, 321–325.
(8) Duimstra, J. A.; Femia, F. J.; Meade, T. J. J. Am. Chem. Soc. 2005,
127, 12847–12855.
* To whom correspondence should be addressed. E-mail: tmeade@
northwestern.edu.
(1) Merbach, A.; Toth, E. The Chemistry of Contrast Agents in Medical
Magnetic Resonance Imaging; John Wiley & Sons, Ltd.: New York,
2001.
(9) Urbanczyk-Pearson, L. M.; Femia, F. J.; Smith, J.; Parigi, G.; Duimstra,
J. A.; Eckermann, A. L.; Luchinat, C.; Meade, T. J. Inorg. Chem.
2008, 47, 56–68.
(10) Urbanczyk-Pearson, L. M.; Meade, T. J. Nat. Protoc. 2008, 3, 341–
350.
(2) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Laufer, R. B. Chem. ReV.
(11) Takeda, A. Brain Res. ReV. 2000, 34, 137–148.
(12) Stefanidou, M.; Maravelias, C.; Dona, A.; Spiliopoulou, C. Arch.
Toxicol. 2006, 80, 1–9.
1999, 99, 2293–2352.
(3) Solomon, I. Phys. ReV. 1955, 99, 559–565.
10788 Inorganic Chemistry, Vol. 47, No. 22, 2008
10.1021/ic801458u CCC: $40.75  2008 American Chemical Society
Published on Web 10/18/2008

ZnII-ActiWated MRI Contrast Agents
Figure 1. Proposed mechanism of the ZnII-activated MRI contrast agent
Gd-daa3.
into the extracellular fluids of the brain has been implicated
in a variety of pathological pathways.14 For example, ZnII
release has been implicated in the precipitation of ꢀ-amyloid
plaques found in Alzheimer’s disease.15,16 The development
of ZnII-activated MRI contrast agents could provide a
valuable tool in the study of in vivo ZnII activity without
using optical agents whose detection is limited by light
scattering and photobleaching.17-19
Figure 2. Complexes 1-4 with systematic variation of the aminoacetate
and pyridyl ZnII-coordinating groups.
Recently, we reported a ZnII-activated MRI contrast agent
where the relaxivity increases upon ZnII binding.20 Our
proposed mechanism suggests that a pair of appended
diaminoacetate arms bind to the GdIII ion to create a
coordinatively saturated complex (q ) 0; Figure 1). In the
presence of ZnII, the diaminoacetates preferentially bind ZnII
and produce a change in the coordination geometry around
the GdIII center, producing an unsaturated complex and
resulting in an increase in relaxivity. On the basis of a
reported crystal structure of a diaminoacetate ZnII-coordina-
tion derivative,21 a distorted square-based pyramidal structure
for ZnII binding to the contrast agent is proposed. Coordina-
tion of ZnII results in the formation of two five-membered
rings sharing the Zn-N bond and can be described as an
intermediate between square-based pyramidal and trigonal-
bipyramidal geometries. The internal rearrangement of the
diaminoacetate binding groups upon binding of ZnII results
in an increase of over 100% in the observed relaxivity of
the agent.
Gd-daa3 is 1.20 This result raised the question concerning
the coordination geometry of the aminoacetate pendant arms
of Gd-daa3 in the presence and absence of ZnII.
Here, we investigate the coordinating groups of a series
of ZnII-activated agents to determine the role of binding and
to prepare agents with a range of ZnII binding (Figure 2).
The systematic variation of GdIII-coordinating aminoacetate
groups and noncoordinating pyridyl groups allowed an
investigation of the coordination geometry of Gd-daa3. We
have discovered that while only one aminoacetate arm is
necessary to efficiently block water to create a coordinatively
saturated GdIII complex, at least two binding groups are
necessary for the binding of ZnII. Therefore, it is possible to
vary one of the aminoacetate arms to effectively tune the
contrast agent to increase the selectivity and sensitivity for
ZnII activation.
Experimental Methods
Gd-daa3 has seven coordinating atoms available from the
macrocyclic chelate and two aminoacetate ligands to bind
GdIII, resulting in a coordination number of nine. Considering
Zn-Gd-daa3, it is possible for one or both aminoacetates to
bind ZnII. If both aminoacetate arms participate in ZnII
binding, two water molecules are expected to bind to the
GdIII ion. However, the experimentally determined q for Zn-
Materials. GdCl3 ·6H2O, EuCl3 ·6H2O, TbCl3 ·6H2O, and 1,4,7,10-
tetraazacyclododecane (cyclen) were purchased and used as is from
Strem Chemicals (Newburyport, MA). Isotopically labeled 1,2-13C-
ethyl bromoacetate was purchased from Cambridge Isotope Labo-
ratories, Inc. (Andover, MA). All other chemicals were purchased
and used as is from Sigma-Aldrich. CH2Cl2, tetrahydrofuran (THF),
and MeCN were dried using a solvent system purchased from Glass
Contour, San Diego, CA. Water was purified using a Millipore
Milli-Q synthesis water system. NMR spectra were recorded on
either Varian (Palo Alto, CA) Inova 400 MHz or Varian Inova
500 MHz instruments with deuterated chloroform or water as
the solvent. Electrospray ionization mass spectrometry (ESI-MS)
spectra were obtained on a Varian 1200 L single-quadrupole mass
spectrometer. Differences in the calculated and found mass
spectrometry data for some complexes are a result of the isotopic
patterns of the GdIII complexes and the 13C-labeled complexes. To
confirm the purity of the final complexes, elemental analysis was
performed by Desert Analytics Laboratory (Tucson, AZ).
(13) Frederickson, C. J.; Koh, J.-Y.; Bush, A. I. Nat. ReV. Neurosci. 2005,
6, 449–462.
(14) Vallee, B. L.; Falchuk, K. H. Physiol. ReV. 1993, 73, 79–118.
(15) Frederickson, C. J.; Cuajungco, M. P.; Frederickson, C. J. J. Alzhe-
imer’s Dis. 2005, 8, 155–160.
(16) Noy, D.; Solomonov, I.; Sinkevich, O.; Arad, T.; Kjaer, K.; Sagi, I.
J. Am. Chem. Soc. 2008, 130, 1376–1383.
(17) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Chem. Soc., Perkin
Trans. 2 2001, 1840–1843.
(18) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Narazaki, M.; Yokawa, T.;
Sakamoto, S.; Yamaguchi, K.; Nagano, T. Chem. Biol. 2002, 9, 1027–
1032.
(19) Zhang, X.-a.; Lovejoy, K. S.; Jasanoff, A.; Lippard, S. J. Proc. Nat.
Acad. Sci., U.S.A. 2007, 104, 10780–10785.
(20) Major, J. L.; Parigi, G.; Luchinat, C.; Meade, T. J. Proc. Nat. Acad.
Sci., U.S.A. 2007, 104, 13881–13886.
(21) Hidalgo, M. A.; Suarez-Varela, J.; Avila-Roson, J. C.; Martin-Ramos,
J. D.; Romerosa, A. Acta Crystallogr. 1996, C52, 810–812.
Synthesis. The synthesis and characterization of Gd-daa3 was
accomplished as previously described.20 Gd-aa3 is a side product
from the synthesis of Gd-daa3 that is collected from the semi-
preparatory high-performance liquid chromatography (HPLC). The
Inorganic Chemistry, Vol. 47, No. 22, 2008 10789

Major et al.
synthetic procedure and characterization for Gd-dpa3 and Gd-apa3
are described. DO3A-tris-tert-butyl ester was synthesized following
literature procedures.22
(400 MHz, CDCl3 w/TMS): δ 6.66 (Ar, 3H), 3.89 (t, J ) 6 Hz,
2H), 3.71 (s, 2H), 3.66 (s, 1H, NH), 3.39-2.26 (multiplicity, 24H),
2.22 (s, 3H), 1.91 (m, 2H), 1.38 (s, 27H), 1.32 (s, 9H). 13C NMR
(400 MHz, CDCl3 w/TMS): δ 173.8, 172.8, 171.4, 170.7, 149.4,
137.1, 128.0, 124.0, 120.7, 109.8, 82.9, 82.6, 81.7, 80.6, 67.1, 57.3,
56.7, 53.9, 51.9, 50.5, 28.1, 26.6, 18.9. MS (ESI+). Calcd for (M
+ H+): m/z 791.5. Found: m/z 792.5. Calcd for (M + Na+): m/z
814.5. Found: m/z 814.5.
1-(3-Bromopropoxy)-3-methyl-2-nitrobenzene (5). To a solu-
tion of 3-methyl-2-nitrophenol (3.0 g, 19.6 mmol) in 200 mL of
dry acetonitrile under nitrogen was added 9.12 g (66.0 mmol) of
anhydrous potassium carbonate. After the reaction turned bright
red because of the deprotonation of the phenol (10 min), 5.96 mL
(58.7 mmol) of 1,3-dibromopropane was added, and the reaction
was allowed to proceed at reflux overnight. After cooling to room
temperature, the reaction was filtered, and the filtrate was washed
with ethyl acetate and concentrated via rotary evaporation. The oily
product was brought up in 100 mL of ethyl acetate and washed
once with water followed by brine. The organic layer was dried
over Na2SO4, filtered, and concentrated. The crude product was
purified on a silica gel column, eluting with 5% ethyl acetate in
hexanes to yield 5 as a light-yellow oil (5.1 g, 95% yield). 1H NMR
(400 MHz, CDCl3w/TMS): δ 7.23 (t, J ) 8 Hz, 1H), 6.85 (d, J )
8 Hz, 1H), 6.81 (d, J ) 7.6 Hz, 1H), 4.12 (t, J ) 5.6 Hz, 2H), 3.48
(t, J ) 6.8 Hz, 2H), 2.22 (s, 3H), 2.21 (m, 2H). 13C NMR (400
MHz, CDCl3 w/TMS): δ 149.9, 142.3, 130.9, 130.8, 123.1, 111.2,
66.8, 32.1, 29.0, 17.1. MS (ESI+). Calcd for (M + H+): m/z 273.0.
Found: m/z 273.9.
({2-Methyl-6-[3-(4,7,10-tris[(tert-butoxycarbonyl)methyl]-
1,4,7,10-tetraazacyclododec-1-yl)propoxy]phenyl}pyridin-2-yl-
methylamino)acetic Acid tert-Butyl Ester (8). In 50 mL of dry
acetonitrile, 0.4 g (0.5 mmol) of 8 was added followed by anhydrous
potassium carbonate (0.28 g, 2.0 mmol) and 2-(bromomethyl)py-
ridine hydrobromide (0.26 g, 1.0 mmol). The reaction was refluxed
overnight and then cooled to room temperature, filtered, and
concentrated via rotary evaporation. The crude product was
absorbed onto silica and purified on a silica gel column, eluting
with 3% methanol in dichloromethane. 8 was collected in 30% yield
(0.13 g, 0.15 mmol) as an orange oil. MS (ESI+). Calcd for (M +
H+): m/z 882.6. Found: m/z 883.2. Calcd for (M + Na+): m/z 905.6.
Found: m/z 905.1.
{4-{3-[2-[Bis(pyridin-2-ylmethyl)amino]-3-methylphenoxy]pro-
pyl}-7,10-bis[(tert-butoxycarbonyl)methyl]-1,4,7,10-tetraazacy-
clododec-1-yl}acetic Acid tert-Butyl Ester (9). Product 6 (0.35
g, 0.5 mmol) was dissolved in methanol and added to a flask
preloaded with 10% palladium on carbon (wet) in catalytic
conditions. The reaction was equipped with a hydrogen reactor at
3.0 bar. After 48 h, the reaction was removed from the hydrogen
reactor and filtered through celite, rinsing several times with
methanol. Reduction of the nitro group to the amine was confirmed
with MS [ESI+; m/z ) 678.5 (M + H+)]. The product was
concentrated and brought up in 30 mL of dry acetonitrile. To this
solution was added anhydrous potassium carbonate (0.17 g, 1.3
mmol) followed by 2-picoyl chloride (0.15 g, 0.9 mmol). The
reaction was refluxed for 5 days to ensure that both picoyl groups
were added. After filtering and rinsing with ethyl acetate and
methanol, the crude product was absorbed onto silica for column
purification, eluting with 5% methanol in dichloromethane. 9 was
{4,7-Bis[(tert-butoxycarbonyl)methyl]-10-[3-(3-methyl-2-nitro-
phenoxy)propyl]-1,4,7,10-tetraazacyclododec-1-yl}acetic Acid
tert-Butyl Ester (6). In a 250 mL round-bottomed flask, 2.0 g (7.3
mmol) of 5 was dissolved in 50 mL of dry acetonitrile. DO3A-
tris-tert-butyl ester (2.5 g, 4.9 mmol) and anhydrous potassium
carbonate (3.4 g, 24.3 mmol) were then added, and the resulting
mixture was refluxed for 2 days. The reaction was cooled to room
temperature and filtered. The crude product was absorbed onto silica
and purified on a silica gel column, eluting with 2% methanol in
dichloromethane. A 66% yield (2.3 g, 3.2 mmol) of 6 was obtained
as a light-yellow oil. 1H NMR (500 MHz, CDCl3 w/TMS): δ 7.24
(t, J ) 8 Hz, 1H), 6.83 (d, J ) 8 Hz, 1H), 6.81 (d, J ) 8 Hz, 1H),
4.01 (t, J ) 6 Hz, 2H), 3.58-2.32 (multiplicity, 24H), 2.23 (s,
3H), 1.92 (m, 2H), 1.38 (s, 27H). 13C NMR (500 MHz, CDCl3
w/TMS): δ 170.6, 170.3, 149.6, 142.0, 131.2, 130.9, 123.2, 111.5,
82.0, 81.8, 66.4, 56.8, 55.5, 53.8, 53.4, 52.6, 50.0, 49.8, 48.0, 28.1,
22.9, 17.0. MS (ESI+). Calcd for (M + H+): m/z 707.5. Found:
m/z 708.5. Calcd for (M + Na+): m/z 730.4. Found: m/z 730.5.
1
obtained as a pale-orange oil (0.15 g, 37% yield). H NMR (500
MHz, CDCl3 w/TMS): δ 8.44 (d, J ) 4.5 Hz, 2H), 7.54 (t, J ) 7.5
Hz, 2H), 7.33 (d, J ) 7.5 Hz, 2H), 7.08 (t, J ) 7 Hz, 2H), 6.95 (t,
J ) 8 Hz, 1H), 6.68 (overlapping doublets, J ) 8 Hz, 2H), 4.35 (s,
2H), 4.30 (s, 2H), 3.91 (t, J ) 6 Hz, 2H), 3.2-2.2 (multiplicity,
24H), 2.17 (s, 3H), 1.95 (m, 2H), 1.42 (s, 27H). 13C NMR (500
MHz, CDCl3 w/TMS): δ 172.8, 160.0, 157.7, 148.9, 138.8, 137.6,
136.2, 126.2, 123.4, 123.0, 121.9, 109.8, 82.8, 82.5, 66.3, 60.2,
55.9, 51.6, 51.1-50.0 (overlapping cyclen peaks), 28.0, 25.6, 19.0.
MS (ESI+). Calcd for (M + H+): m/z 859.6. Found: m/z 860.6.
Calcd for (M + Na+): m/z 882.5. Found: m/z 882.6.
Metalation Procedure. A trifluoroacetic acid (TFA) solution,
95:2.5:2.5 (TFA/ H2O/ triisopropylsilane) was added to the protected
ligands 8 and 9 for 12 h. After TFA was removed by purging the
solution with air and 15 mL of diethyl ether was added, the
precipitate was centrifuged and decanted. The diethyl ether wash
was repeated twice, the final pellet was brought up in H2O, and
the pH was adjusted to 6.5 with 1 M NaOH. A total of 1.1 equiv
of GdCl3 · 6H2O was then added, and the resulting mixture was
stirred at room temperature for several days. Unreacted GdIII
precipitated as Gd(OH)3 after the addition of 1 M NaOH to a pH
of 10 and pelleted. The crude mixture was purified by semiprepra-
tive HPLC on a reverse-phase column, eluting with acetonitrile and
water using an isocratic ramp from 0% to 100% acetonitrile over
35 min. Analytical HPLC-MS was used to confirm the purity and
{2-Methyl-6-[3-(4,7,10-tris[(tert-butoxycarbonyl)methyl]-
1,4,7,10-tetraazacyclododec-1-yl)propoxy]phenylamino}acetic Acid
tert-Butyl Ester (7). Product 6 (1.5 g, 2.1 mmol) was dissolved in
methanol and added to a flask preloaded with 10% palladium on
carbon (wet) in catalytic conditions. The reaction was equipped
with a hydrogen reactor at 3.0 bar. After 48 h, the reaction was
removed from the hydrogen reactor and filtered through celite,
rinsing several times with methanol. Reduction of the nitro group
to the amine was confirmed by MS [ESI+; m/z 678.5 (M + H+)].
The resulting orange oil was dissolved in 25 mL of dry acetonitrile.
To this was added anhydrous potassium carbonate (0.85 g, 6.2
mmol) followed by 0.7 mL (4.5 mmol) of tert-butyl bromoacetate.
The reaction was refluxed for 4 days and followed by thin-layer
chromatography (TLC) to monitor the reaction progression. After
a new spot was observed on the TLC due to the addition of the
second acetate arm, the reaction was cooled to room temperature
and filtered. The crude product was absorbed onto silica and purified
on a silica gel column, eluting with 2% methanol in dichlo-
romethane. 7 was obtained as an orange oil in 25% yield. 1H NMR
(22) Dadabhoy, A.; Faulkner, S.; Sammes, P. G. J. Chem. Soc., Perkin
Trans. 2 2002, 348–357.
10790 Inorganic Chemistry, Vol. 47, No. 22, 2008

ZnII-ActiWated MRI Contrast Agents
identity of the collected fractions. Pure fractions were freeze-dried
and stored in a desiccator. The same procedure was followed with
TbCl3 ·6H2O to obtain the TbIII metal complexes.
methanol, and concentrated via rotary evaporation of the solvent.
The resulting oil was transferred to a 100 mL round-bottomed flask
and dissolved in dry acetonitrile (30 mL). Proton sponge (2.79 g,
13.0 mmol) was dissolved in the reaction mixture before the
addition of 1.0 g (5.92 mmol) of 1,2-13C-ethyl bromoacetate
followed by sodium iodide (1.95 g, 13.0 mmol). After 2 days of
refluxing, the reaction was filtered, rinsed with ethyl acetate, and
concentrated via rotary evaporation. The crude product was
absorbed onto silica and purified on a silica gel column, eluting
with a slow gradient of 2% ethyl acetate in hexanes to 5% ethyl
acetate. The desired product 11 was collected in 75% yield as a
light-yellow oil. 1H NMR (500 MHz, CDCl3 w/TMS): δ 7.00 (t, J
) 8 Hz, 1H), 6.79 (d, J ) 7.5 Hz, 1H), 6.72 (d, J ) 7.5 Hz, 1H),
4.12 (m, 4H), 4.05 (t, J ) 6.5 Hz, 2H), 3.91 (s, broad, 2H), 3.85
(t, J ) 6 Hz, 2H), 3.74 (s, broad, 2H), 2.47 (s, 3H), 2.04 (m, 2H),
1.24 (t, J ) 7 Hz, 6H), 0.91 (s, 9H), 0.07 (s, 6H). 13C NMR (500
MHz, CDCl3 w/TMS): δ 172.5, 157.0, 139.3, 137.7, 126.4, 122.9,
109.7, 64.8, 60.5, 56.6, 49.8, 32.9, 26.2, 21.3, 18.6, 14.4, -5.1.
MS (ESI+). Calcd for (M + H+): m/z 469.3. Found: m/z 472.3.
Calcd for (M + Na+): m/z 492.2. Found: m/z 494.3.
Gd-daa3. Gadolinium(III) Carboxymethyl-{2-methyl-6-[3-
[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]pro-
poxy]phenylamino}acetic Acid (1). Analytical LC-MS showed
a single peak (ESI+). Calcd for (M + H+): m/z 778.2. Found: m/z
781.2. Anal. Calcd for C28H38GdN5O11 ·2H2O·2Na: C, 39.11; H,
4.93; N, 8.14. Found: C, 39.33; H, 4.93; N, 7.84. Tb-daa3.
Analytical LC-MS showed a single peak (ESI+). Calcd for (M +
H+): m/z 779.2. Found: m/z 779.2. Calcd for (M + Na+): m/z 802.2.
Found: m/z 801.2.
Gd-aa3. Gadolinium(III) {2-Methyl-6[3-[4,7,10-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]phenylamino}-
acetic Acid (2). Analytical LC-MS showed a single peak (ESI+).
Calcd for (M + H+): m/z 721.1. Found: m/z 720.2. Anal. Calcd for
C26H38GdN5O9 ·H2O·Na: C, 40.94; H, 5.29; N, 9.18. Found: C,
40.81; H, 5.27; N, 9.02.
Gd-apa3. Gadolinium(III) {{2-Methyl-6-[3-[4,7,10-tris(carboxym-
ethyl)-1,4,7,10-tetraazacyclododec-1-yl]propoxy]phenyl}pyridin-2-
ylmethylamino}acetic Acid (3). Analytical LC-MS showed a single
peak (ESI+). Calcd for (M + H+): m/z 812.2. Found: m/z 813.0.
Anal. Calcd for C32H42GdN6O9 ·2H2O: C, 45.32; H, 5.47; N, 9.91.
Found: C, 45.37; H, 5.79; N, 10.32. Tb-apa3. Analytical LC-MS
showed a single peak (ESI+). Calcd for (M + H+): m/z 813.2.
Found: m/z 814.2.
Gd-dpa3. Gadolinium(III) {4-{3-[2-[Bis(pyridin-2-ylmethyl)-
amino]-3-methylphenoxy]propyl}-7,10-bis(carboxymethyl)-
1,4,7,10-tetraaza-cyclododec-1-yl}acetic Acid (4). Analytical
LC-MS showed a single peak (ESI+). Calcd for (M + H+): m/z
846.3. Found: m/z 847.2. Anal. Calcd for C36H47GdN7O7: C, 51.05;
H, 5.59; N, 11.58. Found: C, 51.15; H, 5.61; N, 11.22. Tb-dpa3.
Analytical LC-MS showed a single peak (ESI+). Calcd for (M +
H+): m/z 847.3. Found: m/z 848.3.
tert-Butyldimethyl-[3-(3-methyl-2-nitrophenoxy)propoxy]si-
lane (10). To a solution of 3-methyl-2-nitrophenol (5.0 g, 32.6
mmol) in dry acetonitrile (300 mL) under nitrogen was added
K2CO3 (11.26 g, 81.5 mmol). After the reaction had turned a bright-
red color because of the deprotonated state of the phenol (about
10 min), (3-bromopropoxy)-tert-butyldimethylsilane (9.04 mL, 39.2
mmol) was added. The reaction was refluxed at 70 °C until it was
a pale-yellow color, cooled to room temperature, and filtered. After
evaporation of the solvent, the mixture was brought up in 100 mL
of ethyl acetate and washed once with an aqueous saturated sodium
bicarbonate solution and once with brine. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was
purified on a silica gel column, eluting with 1% ethyl acetate in
hexanes, yielding 10 as light-yellow crystals after drying (10.11 g,
94% yield). 1H NMR (500 MHz, CDCl3 w/TMS): δ 7.27 (t, J ) 8
Hz, 1H), 6.89 (d, J ) 8.5 Hz, 1H), 6.84 (d, J ) 8 Hz, 1H), 4.15 (t,
J ) 6 Hz, 2H), 3.75 (t, J ) 6 Hz, 2H), 2.30 (s, 3H), 1.96 (m, J )
5.5 Hz, 2H), 0.88 (s, 9H), 0.04 (s, 6H). 13C NMR (500 MHz, CDCl3
w/TMS): δ 150.44, 142.43, 131.06, 130.76, 122.54, 111.01, 65.89,
59.15, 32.23, 26.11, 18.49, 17.17, -5.13, -5.41. MS (ESI+). Calcd
for (M + H+): m/z 325.2. Found: m/z 326.2. Calcd for (M + Na+):
m/z 348.2. Found: m/z 348.1.
{(Ethoxycarbonyl)methyl-[2-(3-hydroxypropoxy)-6-methyl-
phenyl]amino}acetic Acid Ethyl Ester (12). To a solution of 11
(1.1 g, 2.3 mmol) in THF (25 mL) was added tetrabutylammonium
fluoride (1.5 g, 5.8 mmol). After 2 h at room temperature, the
deprotection was completed as observed by TLC (1:3 EtOAc/
hexanes). THF was removed via rotary evaporation. The crude
product was brought up in ethyl acetate and washed once with water
and then brine. The organic layer was dried over sodium sulfate,
filtered, concentrated, and purified through a silica plug, eluting
with 25% ethyl acetate in hexanes and yielding the desired product
1
in 83% yield (0.68 g, 1.9 mmol). H NMR (500 MHz, CDCl3
w/TMS): δ 7.00 (t, J ) 8 Hz, 1H), 6.80 (d, J ) 8 Hz, 1H), 6.74 (d,
J ) 8.5 Hz, 1H), 4.14 (m, 6H, overlapping OCH2CH3 and
OCH2CH2CH2OH), 4.06 (s, broad, 2H), 3.90 (t, J ) 5.5 Hz, 2H),
3.79 (s, broad, 2H), 2.45 (s, 3H), 2.08 (m, 2H), 1.23 (t, J ) 7 Hz,
6H). 13C NMR (500 MHz, CDCl3 w/TMS): δ 172.5, 156.4, 138.5,
137.3, 126.1, 123.4, 110.1, 65.7, 60.2, 49.6, 32.4, 18.7, 14.4. MS
(ESI+). Calcd for (M + H+): m/z 355.2. Found: m/z 358.2. Calcd
for (M + Na+): m/z 378.2. Found: m/z 380.1.
{[2-(3-Bromopropoxy)-6-methylphenyl]ethoxycarbonylmethyl-
amino}acetic Acid Ethyl Ester (13). To a solution of 12 (0.5 g,
1.4 mmol) in dry methylene chloride (15 mL) was added carbon
tetrabromide (0.7 g, 2.1 mmol) followed by the slow addition of
triphenylphosphine (0.7 g, 2.8 mmol). After 2 h at room termpera-
ture, the reaction was washed with water and brine, dried over
sodium sulfate, filtered, and concentrated. The crude product was
purified on a silica gel column, eluting with 5% ethyl acetate in
1
hexanes and giving 13 in 94% yield as an orange oil. H NMR
(500 MHz, CDCl3 w/TMS): δ 7.01 (t, J ) 8 Hz, 1H), 6.81 (d, J )
7 Hz, 1H), 6.73 (d, J ) 8.5 Hz, 1H), 4.11 (m, 6H, overlapping
OCH2CH3 and OCH2CH2CH2Br), 4.00 (s, broad, 2H), 3.73 (s,
broad, 2H), 3.67 (t, J ) 6.5 Hz, 2H), 2.46 (s, 3H), 2.37 (m, 2H),
1.23 (t, J ) 6.5 Hz, 6H). 13C NMR (500 MHz, CDCl3 w/TMS): δ
172.0, 156.5, 139.2, 137.6, 126.3, 123.5, 109.9, 65.7, 60.6, 56.3,
49.6, 32.7, 18.6, 14.5. MS (ESI+). Calcd for (M + H+): m/z 417.1.
Found: m/z 420.0. Calcd for (M + Na+): m/z 440.1. Found: m/z
442.0.
{{2-[3-(tert-Butyldimethylsilanyloxy)propoxy]-6-methylphen-
yl}ethoxycarbonylmethylamino}acetic Acid Ethyl Ester (11). In
30 mL of methanol, 1.0 g of 10 (3.07 mmol) was dissolved and
added to a flask preloaded with 10% palladium on carbon (wet) in
catalytic conditions. The reaction was equipped with a hydrogen
reactor at 3.0 bar for 24 h. Upon completion of reduction, the
reaction was filtered over celite, rinsed several times with 50 mL
{Ethoxycarbonylmethyl-{2-methyl-6-[3-[4,7,10-tris[(tert-bu-
toxycarbonyl)methyl]-1,4,7,10-tetraazacyclododec-1-yl)propoxy]-
phenyl]amino}acetic Acid Ethyl Ester (14). To a solution of
DO3A-tris-tert-butyl ester (0.55 g, 1.1 mmol) in 15 mL of dry
acetonitrile was added anhydrous potassium carbonate (0.44 g, 3.2
mmol). After 5 min, a solution of 13 (0.55 g, 1.3 mmol) in 5 mL
Inorganic Chemistry, Vol. 47, No. 22, 2008 10791

Major et al.
Scheme 1. Synthesis of Gd-apa3 (3) and Gd-dpa3 (4)
of dry acetonitrile was added to the reaction, and the mixture was
refluxed overnight. The reaction was cooled and filtered, rinsing
with acetonitrile followed by methanol. The solvents were removed
by rotary evaporation, and the crude product was purified on a silica
gel column, eluting with 3% MeOH in dichloromethane to yield
14 in 27% yield. After trituration with diethyl ether several times,
a pale-yellow solid was obtained. 1H NMR (400 MHz, CDCl3
w/TMS): δ 6.91 (t, J ) 8 Hz, 1H), 6.72 (d, J ) 7.6 Hz, 1H), 6.62
(d, J ) 8.4 Hz, 1H), 3.95-2.30 (multiplicity, 36H), 2.35 (s, 3H),
1.27 (s, 27H), 1.17 (m, 6H). 13C NMR (500 MHz, CDCl3 w/TMS):
δ 172.6, 164.2, 156.4, 139.0,126.3, 123.3, 109.7, 82.9, 82.6, 66.5,
60.5, 55.8, 51.7, 50.7, 50.0, 49.8, 49.4, 49.2, 47.9, 28.1, 18.4, 14.4.
MS (ESI+). Calcd for (M + Na+): m/z 874.5. Found: m/z 876.6.
Eu-daa3. Europium(III) Carboxymethyl-{2-methyl-6-[3-
[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]propoxy]phenylamino}acetic Acid (15). The protected ligand
14 was first reacted with 1.0 M NaOH for 24 h to deprotect the
ethyl groups. After neutralization to a pH of 7, the crude product
was freeze-dried and then brought up in a solution of TFA for 24 h.
TFA was removed by purging the solution with air, and ap-
proximately 15 mL of diethyl ether was added. The precipitate was
centrifuged and decanted. The diethyl ether wash was repeated two
times, the final pellet was brought up in H2O, and the pH was
adjusted to 6.5 with 1 M NaOH. A total of 1.1 equiv of EuCl3 ·6H2O
was added and stirred at room temperature for several days.
Unreacted EuIII precipitated as Eu(OH)3 after the addition of 1 M
NaOH, and the crude mixture was purified by semipreprative HPLC,
eluting with acetonitrile and water. Analytical HPLC-MS was used
to confirm the purity and identity of the collected fractions and
showed a single peak with MS (ESI+). Calcd for (M + H+): m/z
772.6. Found: m/z 772.5.
pulse sequence with the appropriate recycle delays. The spectrom-
eter fits 10 data points to an exponential decay for each T1
measurement and calculates the standard deviation for each T1,
representing an error of less than 1%. This titration was repeated
until a 1:3 ([Gd]:[Zn]) ratio was reached.
Luminescence Lifetime Measurements. The fluorescence decay
rates of the terbium analogues of 1, 3, and 4 in buffered H2O and
D2O were measured on a Hitachi F4500 fluorometer monitoring
the emission at 544 nm with an excitation of 254 nm. Aliquots of
HEPES buffer and ZnCl2 in HEPES were freeze-dried before being
brought up in D2O to ensure there was no water present. A 200
µM solution of the terbium complex in HEPES buffer was measured
in the presence of 300 µM ZnCl2 and without ZnCl2. A total of 25
scans were averaged and fit to a monoexponential decay with an
r2 value of 0.99.
Inductively Coupled Plasma Atomic Emission Spectrometry
(ICP-AES). The concentration of each sample for relaxivity was
determined by ICP-AES (Varian). A 10 µL aliquot of each sample
was digested in 90 µL of nitric acid. Each sample was diluted with
water to the appropriate concentration for ICP-AES analysis.
Gadolinium concentrations were determined from a standardized
curve of six standards ranging from 0 to 500 ppb GdIII, measuring
the emission of GdIII at 335.048, 336.224, and 342.246 nm.
Results
Synthesis. The syntheses of 3 and 4 are outlined in
Scheme 1. Starting with 3-methyl-2-nitrophenol, 1,3-dibro-
mopropane was added using potassium carbonate in dry
acetonitrile. DO3A-tris-tert-butyl ester was synthesized fol-
lowing literature procedures22 and combined with 5 under
basic conditions to yield 6. The nitro group of 6 was then
reduced under standard palladium-catalyzed hydrogenation
conditions and reacted with either tert-butyl bromoacetate
to yield 7 or with 2-picoyl chloride to yield 9. The final
pyridyl binding group was added to 7 using 2-bromopyridine
hydrobromide to yield the final tert-butyl-protected ligand
8. Both 8 and 9 were reacted with trifluoroacetic acid to
Relaxivity Measurements. A 1 mM solution of each GdIII
complex was prepared in buffer containing 100 mM KCl/100 mM
HEPES at pH 7.4. These were serially diluted four times to give
500 µL of five different sample concentrations at a [Gd]:[Zn] ratio
of 1:0. Aliquots of a 5.0 mM ZnCl2 solution in HEPES were added
to each of the samples to give a [Gd]:[Zn] ratio of 1:0.5. After 30
min of incubation at 37 °C, T1 measurements were performed on
a Bruker mq60 minispec relaxometer with an inversion-recovery
10792 Inorganic Chemistry, Vol. 47, No. 22, 2008

ZnII-ActiWated MRI Contrast Agents
Scheme 2. Synthesis of 13C-Isotopically Labeled Eu-daa3 (15; * ) 13C)
Table 1. Relaxivity in the Presence and Absence of ZnII at 60 MHz
and 37 °C in HEPES Buffer
remove the tert-butyl protecting groups and reacted with
GdCl3 ·6H2O at a pH of 6.5 for 2 days. The final compounds
3 and 4 were purified by semipreparatory HPLC and
characterized by LC-MS and elemental analysis.
r1 (mM-1s-1
)
0 equiv of ZnII
1 equiv of ZnII
% change in r1
The 13C isotopic labeling of the aminocarboxylates of Gd-
daa3 was synthesized following previously published pro-
cedures with some modifications outlined in Scheme 2.20
The synthesis begins with alkylation of the commercially
available 3-methyl-2-nitrophenol with the tert-butyldimeth-
ylsilyl-protected alcohol to give 10 in 95% yield. After
standard hydrogenation conditions, the two aminoacetate
arms were added using 1,2-13C-ethyl bromoacetate in the
presence of proton sponge and sodium iodide. Deprotection
of the TBDMS protecting group was achieved with tetrabu-
tylammonium fluoride to yield 12, which was brominated
with carbon tetrabromide in the presence of triphenylphos-
phine to give 13. The addition of DO3A-tris-tert-butyl ester
was achieved with potassium carbonate in acetonitrile under
refluxing conditions to give the fully protected ligand 14.
The ethyl groups were deprotected through the addition of
1 M NaOH at room temperature. After neutralization, the
tert-butyl groups were deprotected with TFA before the
addition of the metal with EuCl3 ·6H2O. The final compound
was purified by semipreparatory HPLC and characterized by
analytical LC-MS.
Relaxivity. To evaluate the binding properties of the
aminoacetate and pyridyl groups employed in complexes
1-4, relaxivities were first measured in the absence of ZnII
at 60 MHz and 37 °C in HEPES buffer to determine the
effectiveness in creating a coordinatively saturated GdIII
complex with low relaxivity. As seen in Table 1, the
complexes with one or two aminoacetates are binding to GdIII
and thus create a low-relaxivity complex. The relaxivity of
Gd-dpa3 of 7.5 mM-1 s-1 illustrates the inability of the
pyridyl nitrogen atoms to bind with GdIII and therefore not
reduce water access to the GdIII ion. The lowest relaxivity
observed is for Gd-daa3, suggesting that both aminoacetates
participate in the binding of GdIII, as depicted in Figure 1.
The two complexes with only one aminoacetate, Gd-aa3 and
Gd-daa3
Gd-aa3
Gd-apa3
Gd-dpa3
2.3
4.1
3.4
7.5
5.1
4.2
6.9
7.2
+ 121%
+ 2.4%
+ 103%
- 4.0%
Gd-apa3, do not have as low of a relaxivity as that observed
for Gd-daa3; however, they still have the ability to bind
GdIII and effectively reduce the access of water. This suggests
that a coordination number of eight is sufficient in creating
a coordinatively saturated GdIII complex. The lower relaxivity
for Gd-apa3 compared to Gd-aa3 may be due to the added
steric effects in reducing water access from the second
pyridyl arm of Gd-apa3. While there is no direct evidence
from these experiments of the effect on second-sphere
solvation, the presence of the noncoordinating pyridyl ligand
may allow for the reduction of second-sphere water mol-
ecules, resulting in an overall decrease in the observed
relaxivity.
In the presence of 1 equiv of ZnCl2 in HEPES buffer at
60 MHz and 37 °C, the relaxivities of both Gd-daa3 and
Gd-apa3 increase by 121% and 103%, respectively, while
there was no significant change in the relaxivity for Gd-aa3
(Table 1). Importantly, Gd-aa3 exhibits no increase in r1
upon the addition of ZnII, implying that there is no change
in the coordination environment. This is the only complex
with only one available binding group and, therefore, it is
reasonable to suggest that two binding groups are required
for coordinating ZnII. Given that Gd-dpa3 was not able to
create a coordinatively saturated structure when no ZnII was
present, it was expected that there would be little change in
its relaxivity with the addition of ZnII, as observed.
Luminescence. The relaxivity studies presented suggest
that, in the absence of ZnII, complexes with at least one
available aminoacetate arm to coordinate GdIII have low
relaxivity values, as would be expected for a q ) 0 complex.
While the relaxivity measurements reflect the effect of both
Inorganic Chemistry, Vol. 47, No. 22, 2008 10793

Major et al.
Table 2. Fluorescence Decay Lifetimes of 1, 3, and 4 in H2O and D2O
and Their Calculated q Values
τH O (ms) + τD O (ms) +
2
2
τH O (ms) τD O (ms)
q
ZnII
ZnII
q + ZnII
2
2
1
3
4
1.97
1.76
1.21
2.71
2.21
2.33
0.3
0.2
1.4
1.46
1.63
1.18
2.65
2.67
2.33
1.0
0.8
1.5
the inner-sphere water molecules and the second solvation
shell, fluorescence lifetime measurements can directly report
on the first solvation sphere. To determine if, in fact, an eight-
coordinate complex could provide a coordinatively saturated
complex, the hydration numbers (q) of the terbium analogues
of 1, 3, and 4 were determined using time-based fluorescence
microscopy measurements. The fluorescence decay rates of
the TbIII analogues in water and D2O with and without ZnII
present were measured to calculate q. Because of the efficient
vibronic coupling of the TbIII excited state to the O-H
oscillators compared to that of the O-D oscillators, a shorter
luminescence lifetime is observed in H2O than in D2O.23 The
number of coordinated water molecules is then calculated
using eq 1.24,25 The fluorescence lifetimes and calculated q
values are summarized in Table 2. These measurements
confirm a change in the coordination environment for Gd-
daa3 and Gd-apa3 from a q ) 0 complex to a q ) 1
complex. The hydration numbers for Gd-apa3 provide
further evidence of an eight-coordination GdIII complex. In
the absence of ZnII, the eight coordinating arms bind GdIII
with no open site for water access, while in the presence of
ZnII, there is one inner-sphere water molecule bound to GdIII
along with the seven coordinating groups from the chelate.
The hydration numbers calculated for Gd-dpa3 confirm that
in the absence of ZnII the pyridyl groups do not bind GdIII.
Therefore, the hydration number in the absence and presence
of ZnII remains unchanged.
Figure 3. (A) 13C NMR in the absence of ZnII of Eu-daa3 and (B) 13C
NMR in the presence of 1 equiv of ZnII with Eu-daa3 providing evidence
of the interaction of the aminoacetate groups with ZnII and EuIII.
direct evidence of the interaction of the aminoacetates with
the ZnII and EuIIImetal centers.
1
1
Discussion
q ) 4.2 ms
-
- 0.06
(1)
(
[
τH O) (τD O
)
]
2
2
The four complexes investigated in this study provide
evidence of the mechanism of activation for ZnII-activated
contrast agents. The first of this series of ZnII-activated
contrast agents, Gd-daa3, has two aminoacetate arms that
are proposed to vary metal-binding coordination upon the
addition of ZnII.20 Three new complexes were synthesized
in which the ZnII binding groups are modified with one or
two pyridyl groups (Gd-apa3 and Gd-dpa3, respectively)
or with the complete removal of one of the coordinating
aminoacetate arms (Gd-aa3). Investigation of the relaxivities
and q values determined that two structural requirements
were necessary for the preparation of ZnII-activated MRI
agents. The first requirement is the presence of at least one
aminoacetate to create a coordinatively saturated complex
before binding of ZnII. The second requirement is the need
for at least two ZnII coordination groups to effectively bind
ZnII and create a high-relaxivity state when ZnII is present.
Evidence of the efficient reduction of water access with
only one aminoacetate coordinating arm present is demon-
strated by the low-relaxivity values of complexes 1-3 in
the absence of ZnII. The hydration numbers of Tb-daa3 and
Europium 13C NMR Spectroscopy. In order to investi-
gate the ZnII binding capability of the aminoacetate arms,
the EuIII analogue of Gd-daa3 was synthesized with 13C
isotopic labeling of the aminoacetate groups. In the absence
of ZnII, no carbon shifts are visible because of the line-
broadening effects of the paramagnetic EuIII metal center
when the aminoacetate arms are bound to EuIII (Figure 3A).
Upon the addition of ZnII to the same sample, two peaks are
seen in the 13C NMR spectrum corresponding to the two
carbon atoms on the aminocetates with shifts at 178 ppm
for the carbonyl and 62 ppm for the methylene (Figure 3B).
When bound to ZnII and not bound directly to the EuIII metal,
the line broadening due to the paramagnetism is reduced,
resulting in two carbon shifts from the 13C-labeled aminoac-
etates that were not previously seen. These results provide
(23) Kropp, J. L.; Windsor, M. W. J. Chem. Phys. 1965, 42, 1599–1608.
(24) Horrocks, W. D.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384–
392.
(25) Quici, S.; Cavazzini, M.; Marzanni, G.; Accorsi, G.; Armaroli, N.;
Ventura, B.; Barigelletti, F. Inorg. Chem. 2005, 44, 529–537.
10794 Inorganic Chemistry, Vol. 47, No. 22, 2008

ZnII-ActiWated MRI Contrast Agents
Tb-apa3 before the addition of ZnII indicate a coordinatively
saturated q ) 0 complex. However, the high-relaxivity value
of Gd-dpa3 in the absence of ZnII and the hydration number
of Tb-dpa3 are indicative of a coordinatively unsaturated
structure in which water can access the GdIII metal center,
providing further evidence of the need for one aminoacetate
to create a low-relaxivity complex. The slight decrease of
relaxivity observed for Gd-dpa3 in the presence of ZnII may
be due to a more rigid conformation that reduces the access
of water to GdIII. While the hydration numbers remain
statistically unchanged for Gd-dpa3 in the presence and
absence of ZnII, the observed decrease in relaxivity could
be interpreted as a change in the second-shell solvation
environment.
The second criterion is that ZnII binding can only occur if
there are at least two ZnII-coordinating arms present. The
addition of ZnII increases the relaxivity by more than 100%
for Gd-daa3 and Gd-apa3. In the case of Gd-aa3, only one
coordinating arm is available for ZnII binding and can
therefore not coordinate ZnII to create a change in relaxivity
in the presence of ZnII. NMR spectroscopy of 13C-labeled
aminoacetates of Eu-daa3 provides evidence of the ZnII
coordination to the aminoacetates to support the mechanism
of activation presented in Figure 1, where a change in the
coordination number from nine to eight is observed upon
ZnII binding.
A thorough understanding of the mechanism of ZnII
activation of the MRI contrast agent Gd-daa3 described in
this work is important for the development of new agents
with a range of binding constants for ZnII. We have
determined that one of the aminoacetate arms can be
modified with a variety of functional groups while still
maintaining the ability to modulate q, which is necessary
for activation of the contrast agent.
Acknowledgment. This work was supported by the
National Institutes of Health/National Institute of Biomedical
Imaging and Bioengineering Grant 1 R01 EB005866-01 and
the Nanomaterials for Cancer Diagnostics and Therapeutics
under Grant 5 U54 CA1193 41-02. J.L.M. is a Scholar of
the Chicago Chapter of the ARCS (Achievement Rewards
for College Scientists) Foundation. R.M.B. received an NSEC
undergraduate research fellowship for support of this work.
IC801458U
Inorganic Chemistry, Vol. 47, No. 22, 2008 10795