A modular system for the synthesis of multiplexed magnetic resonance probes

Article abstract of DOI:10.1021/ja1099616

We have developed a modular architecture for preparing high-relaxivity multiplexed probes utilizing click chemistry. Our system incorporates azide bearing Gd(III) chelates and a trialkyne scaffold with a functional group for subsequent modification. In op

Full text of DOI:10.1021/ja1099616

ARTICLE
pubs.acs.org/JACS
A Modular System for the Synthesis of Multiplexed Magnetic
Resonance Probes
Daniel J. Mastarone,† Victoria S. R. Harrison,† Amanda L. Eckermann,† Giacomo Parigi,‡ Claudio Luchinat,‡
and Thomas J. Meade*,†
†Department of Chemistry, Molecular Biosciences, Neurobiology and Physiology, and Radiology, Northwestern University,
2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
‡CERM and Department of Chemistry, University of Florence, via L. Sacconi 6, 50019 Sesto Florence, Italy
S Supporting Information
b
ABSTRACT: We have developed a modular architecture for
preparing high-relaxivity multiplexed probes utilizing click
chemistry. Our system incorporates azide bearing Gd(III)
chelates and a trialkyne scaffold with a functional group for
subsequent modification. In optimizing the relaxivity of this new
complex, we undertook a study of the linker length between a
chelate and the scaffold to determine its effect on relaxivity. The
results show a strong dependence on flexibility between the
individual chelates and the scaffold with decreasing linker length leading to significant increases in relaxivity. Nuclear magnetic
resonance dispersion (NMRD) spectra were obtained to confirm a 10-fold increase in the rotational correlation time from 0.049 to
0.60 ns at 310 K. We have additionally obtained a crystal structure demonstrating that modification with an azide does not impact the
coordination of the lanthanide. The resulting multinuclear center has a 500% increase in per Gd (or ionic) relaxivity at 1.41 T versus
small molecule contrast agents and a 170% increase in relaxivity at 9.4 T.
’ INTRODUCTION
water residence lifetime, and T1e the electronic relaxation time.
By attaching a small molecule CA to a macromolecule (e.g.,
protein, polymer, nanoparticle, viral capsid), the molecular
tumbling decreases resulting in a longer τR and a subsequent
increases in relaxivity.7,25-32 Although macromolecular systems
have shown significant promise as high relaxivity CAs at low field,
they are difficult to characterize and are frequently poly-
disperse.31,33 For macromolecular CAs, an increase in relaxivity
is observed between 0.5 and 1.5 T due to the field dependence of
T1e. The increase in relaxivity is due to the T1e contribution to τc1
resulting from the long τR of the macromolecular CAs. However,
this increase is followed by a rapid decrease in relaxivity as the
field strength increases beyond 1.5 T. This effect results from the
long τc1 value which shifts the onset of the relaxation dispersion
to smaller proton Larmor frequencies.34
Magnetic resonance imaging (MRI) is a widely used modality
in experimental research and clinical diagnostics.2 The intrinsic
contrast of MR images can be augmented by the use of MR
contrast agents (CAs) that are derived from two primary classes:
superparamagnetic particles3-5 and paramagnetic chelates.6 The
relaxivity (mM-1 s-1) of a paramagnetic contrast agent reflects
its ability to shorten the T1 relaxation time of water which results
in a brighter MR image. The higher the relaxivity, the more
sensitive the agent.6,7
During the past decade, there has been a surge in the
development of new gadolinium-based MR contrast agents.
These efforts have focused on signal amplification, targeting,
and bioactivated or responsive probes.7-22 Recently, multimodal
agents have emerged that enhance MR contrast and simulta-
neously provide the ability to image the target with other
techniques (fluorescence microscopy or positron emission
tomography)12,23 for in vivo targeting and fate mapping of
cells.8,22
The relaxivity of MR contrast agents depends heavily on
magnetic field strength.24 At low magnetic fields (1.5-3 T),
the relaxivity of small molecule contrast agents are limited by
their short rotational correlation time (τR).7 As shown in eq 1 an
optimal relaxivity occurs when the correlation time (τc1) of the
Gd(III) CA is equal to the inverse of the Larmor frequency (1/ωI)
of the proton. This correlation time contains contributions from
three process, τR the rotational correlation time, τm the mean
1
1
1
1
1
opt
c1
τ
¼
;
¼
þ þ
τR τm T1e
ð1Þ
ωI τc1
The need for increased sensitivity for molecular and cellular
imaging in research laboratories has driven the development of
high field MR systems.24,35 Caravan and co-workers demon-
strated that as field strength increases the optimal value for τR
decreases, from ≈20 ns for low field to ≈0.5 ns for high field
magnets.24
Received: November 5, 2010
Published: March 17, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Contrast Agent 3, from the Trialkyne Core 2, and Gd(III) Contrast Agent 1 through Click Chemistry
ARTICLE
To address the need for contrast agents that can operate
effectively in the high field MR instruments and simultaneously
provide the option to install multiple modalities, the develop-
ment of a synthetically flexible contrast agent core is required.
Toward this goal, we have designed a new class of contrast agent
conjugates that are modular, monodisperse, exhibit high relax-
ivity, and can be subsequently modified (Scheme 1).
rigidity.22 Here, we describe three new azide-functionalized
chelates and a new phenol-based scaffold with three alkyne
groups to take advantage of facile click chemistry. The alcohol
functional group on the phenol scaffold is orthogonal to click
chemistry and can be used in subsequent modification.
The new macrocyclic chelates 1, 4, and 5 were designed to
investigate the effect of linker length on relaxivity (Figure 1).
Preparation begins with a ring-opening of epichlorohydrin with
sodium azide to provide 1-azido-3-chloropropan-2-ol 6. Addition
of 6 to the trisubstituted macrocycle (tris-t-butyl-DO3A) pro-
vides the protected ligand 7. Deprotection of the t-butyl esters
with formic acid at 50 °C followed by metalation provides
contrast agent 1 after HPLC purification (Scheme 2). Chelates
4 and 5 were prepared from tris-t-butyl-DO3A-monoacetic acid
and N3(CH2)nNH2 through peptide coupling where n = 2, 3 see
(Scheme 1, Supporting Information). These chelates were
metalated using Gd(OAc)3 and purified using reverse phase
HPLC.
Complex 1 was characterized by X-ray crystallographic anal-
ysis (v:v:v, 1:1:1, mixture of water, acetonitrile, and acetone) and
revealed no significant differences between the chelate structure
of 1 and [Gd(HP-DO3A)(H2O)] (Figure 2).50 The O8-Gd
bond length is consistent with that reported for [Gd(HP-
DO3A)(H2O)] (ProHance) indicating that the overall structure
is not perturbed by the addition of the azide at C16 and the
coordination geometry of 1 is similar to [Gd(HP-DO3A)-
(H2O)].
’ RESULTS AND DISCUSSION
Several examples of multimeric conjugates with intermediate
τR values between (0.5 and 4 ns) exist in the literature.22,36 For
example, the work of Martin and co-workers is an early example
of a modular agent that has been modified with a fatty acid to
bind to bovine serum albumin (BSA).37 Ranganathan and co-
workers have found a 25% increase in relaxivity when the number
of freely rotating atoms in their linker was decreased.38 Addi-
tionally, a 40% increase in relaxivity was seen by Henig and co-
workers in their silsesquioxane contrast agents by changing from
an ethylene to a benzene linker.39 The multimeric complexes of
Livramento and Kotkovꢀa demonstrate very high relaxivities at 20
and 60 MHz due to increased size and hydration number.40-43
These last two examples have shown promise as high field agents
but do not possess further functionality for targeted or multi-
modal imaging projects. The properties of these agents are
compared in Table 1. In related work with macromolecular
agents, Zhang and Nair use peptide tetramers designed to bind to
human serum albumin (HSA) or fibrin and demonstrate that
multilocus binding results in an increase in relaxivity due to
increased rigidity.44-46 Avedano and Datta show that increasing
the rigidity of their linkers (alkyl vs aromatic) to HSA and viral
capsids respectively increased relaxivity by more than 50%.29,47
The works of Rudovsky and co-workers demonstrate the im-
portance of internal motion in dendritic systems by utilizing ion
paring between the PAMAM dendrimer and a polyarginine to
reduce internal motion and increase relaxivity.48,49
Preparation of scaffold 2 is shown in Scheme 3. The synthesis
begins with the alkylation of tribromophenol with ethyl 5-bro-
movalerate in DMF with K2CO3 overnight at 50 °C to form 8 in
high yield. A Sonogashira reaction using 8 and trimethylsilyl
(TMS) acetylene provides the protected trialkyne 9. The TMS
groups are removed using KF in ethanol and the ethyl ester is
saponified in dioxane/water with NaOH to form the desired
scaffold 2 (Scheme 3). This scaffold includes a carboxylic acid
that can be functionalized before or after the click chemistry to
form the final conjugate.
We have previously demonstrated that the triazole linkages
formed by click chemistry are effective in generating multimeric
CAs with relatively high relaxivities as a result of increased linker
A series of agents (3, 11, 12) were synthesized with 1, 3, or 4
methylene groups between the scaffold and the chelate. Click
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Table 1. Summary of Multimeric Contrast Agentsa
ref
field strength MHz
solution
temperature °C
q
per gd relaxivity mM-1 s-1 no. of Gd(III) centers molecular relaxivity mM-1 s-1
this workb
this workb
this workb
Zhangb
60
60
60
64
64
60
60
60
60
24
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
DPBS
37
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
15.4
8.9
3
3
3
4
4
3
6
7
1
4
4
4
4
4
4
4
2
2
6.9
3
6
8
4
2
8
8
1
46.2
26.7
21.9
50
water
37
water
37
7.3
TBS
35
12.5
18
Zhangb
TBS/fibrin
water
35
72
Song
37
5.9
17.7
65.8
85.4
3
Song
water
37
11
Song
water
37
12.2
3
Gd(HP-DO3A)
Jebasingh
Zhangb
Zhangb
Nairb
Nairb
Martinb
Martinb
Martinb
MOPS
Tris pH 5.6
PBS
37
35
7
28.1
41.2
156.4
82.4
110.8
41.6
98.4
9.4
37
10.3
39.1
20.6
27.7
10.4
24.6
4.7
PBS/HSA
TBS
37
37
TBS/fibrin
water
37
N/A
N/A
N/A
N/A
25
water/BSA
water
Martinb
water/BSA
water
9.3
18.6
149
Kotkovꢀac
Livramento
Livramento
Ranganathan
Ranganathan
Ranganathan
Henig
21.6
15.7
20.1
13.0
9.8
water
37
47.1
120.6
104
water
37
water
40
water
40
39.2
10.8
96.8
136.8
4.5
water
40
5.4
water
25
12.1
17.1
4.5
Henig
water
25
Gd(DTPA)
water
N/A
a The contrast agents in this table are divided based on the field strength of relaxivity measurements. The differences in field strengths and temperature
used in collecting this relaxivity data make it difficult to compare to the molecules in our current work. In general relaxivity decreases from 20 to 60 MHz,
and relaxivity also decreases with increasing temperature. b Bifunctional. c Multimodal.
Scheme 2. Synthesis of Complex 1
Figure 1. Macrocyclic chelates with increasing linker length.
chemistry was used to conjugate 1, 4, and 5 to scaffold 2 using
standard conditions resulting in an observable correlation between
relaxivity and linker length. The longest linker (12) has the lowest
relaxivity (7.3 mM-1 s-1 at 1.41 T), while the shortest linker (3)
exhibits the highest relaxivity (15.4 mM-1 s-1 at 1.41 T)
hydration number q in solution. Fluorescence analysis of
[Eu(HPN3DO3A)(H2O)] showed a value of 0.90 ( 0.1. Fol-
lowing attachment of [Eu(HPN3DO3A)(H2O)] to scaffold 2
the hydration number was maintained at 0.87 ( 0.1. These
results confirm that the addition of the azide and the subsequent
click chemistry do not affect the hydration number. The relax-
ivities of 1 and 3 were measured at 1.41 T and 1 is similar to
[Gd(HP-DO3A)(H2O)](Table 2). At this low field strength, the
per Gd relaxivity of 3 is 5-fold higher than that of 1.
(Figure 3). This result is not surprising considering that increasing
the rigidity of the linker between the scaffold and the individual
chelates limits local rotation. This effect has been shown to
increase relaxivity in other systems.22,26,38 It should be noted that
τm could be an additional limitation to the relaxivity of (11) and
(12) due to the monoamide ligand structure of these chelates.
[Eu(HPN3DO3A)(H2O)] was synthesized to confirm that
the addition of the azide to the structure did not perturb the
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Figure 2. (a) 2D structures of [ Gd(HPN3DO3A)(H2O)] (1) and [Gd(HPDO3A)(H2O)] (b) Thermal ellipsoid plot of (1) drawn at 35% probability
level. Hydrogen atoms are omitted for clarity. Distances (Å): Gd1-O1 = 2.47; Gd1-O2 = 2.37; Gd1-O8 = 2.43. The differences between these values
and the corresponding values for [Gd(HP-DO3A)(H2O)] are 0.03, 0.01, 0.01, respectively.
Scheme 3. Alkyne Scaffold 2 Designed for Orthogonal Modification through Click Chemistry with 1 and Peptide Coupling
To observe the effect of field strength on the relaxivities of 1
and 3, these values were measured at 9.4 T. At this field strength,
the relaxivity of 1 is only 10% lower than at 1.41 T, while 3 shows
a 70% decrease. This effect is typical of small molecule chelates
such as 1 with short τR values. In these cases, the relaxation rates
are relatively unaffected by field strength. The pronounced
decrease in relaxivity of 3 at higher field strength suggests the
complex has a long τR. The per Gd relaxivity of 3 at 9.4 T is 170%
that of 1. By controlling the size of the CA, τR can be regulated to
achieve a significant increase in relaxivity.
0.0002-0.95 T) for 1 and 3 at 298 and 310 K (Figure 4). The
nuclear magnetic resonance dispersion (NMRD) profiles35 of 1
exhibit a single dispersion. This result is ascribed to one water
molecule coordinated to the paramagnetic Gd(III) ion, the protons
of which are dipolarly coupled to the electron spins with a correlation
time corresponding to the tumbling time of a small molecule, and the
additional contribution of diffusing water molecules. The relaxivity of
3 is (i) much larger than that of 1 and (ii) exhibits a dispersion
occurring at smaller frequencies. Both features indicate that the
correlation time τc1 is sizably increased.51 A peak in relaxivity appears
in the high frequency region, indicating that the correlation time is
field dependent and must be affected by the electron relaxation time
Τ1e. In turn, this result means that for 3, τR is longer than Τ1e at least
To obtain a better estimate of the increase in the τR between
compounds 1 and 3, water proton relaxivity was measured from 0.01
to 40 MHz proton Larmor frequency (corresponding to fields of
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Figure 3. As the linker length between the azide and the chelate increases (left to right) the relaxivities (at 1.41 T and 310 K) decrease. This effect is
attributed to a reduction in the local rotation of the chelates for the shorter linker lengths.
Table 2. Relaxivities of Compounds 1 and 3 at 1.41 and 9.4 T at 37 °C
field
1.41 T
9.4 T
per Gd(III)
molecular
per Gd(III)
molecular
[Gd(HP-DO3A)(H2O)]
2.99 ( 0.5a
3.05 ( 0.06b
15.4 ( 0.8b
2.99 ( 05a
3.05 ( 0.06b
46.2 ( 2.4b
1 (mM-1 s-1
3 (mM-1 s-1
)
2.79 ( 0.04b
4.8 ( 0.3b
2.79 ( 0.04b
14.4 ( 0.9b
)
a 10 mM MOPS, 100 mM NaCl, pH 7.4, 37 °C.1 b DPBS 10 mM, pH 7.4, 37 °C.
at low fields, so that at such fields Τ1e dominates the correlation time
τc1. Τ1e increases with increasing magnetic field strength, and
therefore the correlation time τc1 is determined by both τR and
Τ1e in the high field region of Figure 4. The temperature dependence
observed in the relaxivity profiles of both complexes indicates that
water molecules are in the fast exchange regime. Therefore, their τm
is not strongly limiting the relaxivity.
The profiles for 1 have been fit (solid lines in Figure 4) using
the standard Solomon-Bloembergen-Morgan (SMB) model,52,53
that is, neglecting the presence of static zero-field splitting (ZFS),
and the Freed equation.54,55 These models calculate the inner-
sphere and the outer-sphere contributions, respectively. The
SBM model describes the relaxation profiles from the number
of coordinated water molecules and their distance from the
unpaired electrons, and from several dynamic parameters, that
are the molecular reorientation time, the electron relaxation time,
and the lifetime of coordinated water protons. The Freed
equation describes the contribution to relaxation due to water
molecules freely diffusing around the paramagnetic complex
through the diffusion constant, the distance of closest approach,
and the electron relaxation time. We assume that the two protons
of the coordinated water molecule are located at 3.1 Å from the
metal ion, with lifetimes τm of 350 and 300 ns at 298 and 310 K
respectively, as previously found for [Gd(HP-DO3A)(H2O)] at
298 K.56 Diffusion water molecules were considered with stan-
dard values for the diffusion constants of 2.5 ꢀ 10-5 and 3.5 ꢀ
10-5 cm2 s-1 at 298 and 310 K,51 respectively, and a distance
of closest approach of 3.6 Å. The best fit parameters were the
Figure 4. Water proton relaxativity data for complexes 1 (b, O) and 3
(2, Δ) at 298 K (solid symbols) and 310 K (open symbols). The solid
lines are the best-fit curves (see text).
field-dependent electron relaxation time, Τ1e, described by the
transient ZFS Δt and the electron correlation time τv, and the
tumbling time τR. The calculated best fit values were 0.027 (
0.003 cm-1 for Δt, 28 ( 2 and 26 ( 3 ps for τv at 298 and 310 K,
respectively, and 0.067 ( 0.004 and 0.049 ( 0.004 ns for τR at
298 and 310 K, respectively. These values correspond well to
those previously obtained for similar small Gd(III) complexes by
analyzing the relaxation data using the same model.6
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Table 3. Rotational Correlation Times of Compounds 1 and
3 at 298 and 310 K
τR
298 K
310 K
1
3
0.067 ( 0.004 nsa
0.74 ( 0.03 nsa
0.049 ( 0.004 nsa
0.60 ( 0.02 nsa
a Gd(III) proton distance 3.1 Å, τM of 350 and 300 ns at 298 and 310 K,
diffusion rate of water 2.5 ꢀ 105 and 3.5 ꢀ 105 cm2s-1 at 298 and 310 K.
Figure 5. MR images of 3 and 1 at 7 T using a RARE pulse sequence
with a TE/TR of 11/200 ms. The molecular concentrations of 3 and 1
range from 60 μM to 7.5 μM in 10 mM DPBS. The Gd(III) ionic
concentration is three times higher for compound 3 than for 1 based on
the molecular structure.
The profiles for 3 could not be satisfactorily fit using the SBM
model presumably due to the effect of a static ZFS in the presence
of a slower molecular tumbling. The profiles were fit according to
the “modified Florence” approach53,57-59 to obtain estimates of
the parameters on which the relaxivity depends. In such an
approach, the effect of static ZFS on the splitting of the energy
levels of all spin states is considered for the calculation of the
nuclear spectral densities, provided that τR is much larger than
Τ1e. The solid lines in Figure 4 are the best-fit curves obtained for
the two protons of the coordinated water molecule at 3.1 Å from
the metal ion with a τR of 0.74 ( 0.03 ns and of 0.60 ( 0.02 ns at
298 and 310 K, respectively. All other parameters have the same
values used in the analysis performed with the SBM and Freed
concentration. This data demonstrate the improved performance
of 3 vs 1 at high field strength which results in significant contrast
enhancement.
In conclusion, we have developed a multiplexed and modular
MR contrast agent scaffold with a 500% increase in per Gd
relaxivity at 1.41 T and a 170% increase at 9.4 T versus small
molecule MR agents. Using this approach, we are preparing
scaffolds that support multiple CAs while simultaneously pro-
viding the ability to incorporate functional groups for sub-
sequent modification with targeting ligands, fluorophores, and
nanoparticles.
models, except for the transient ZFS Δt of 0.018 ( 0.001 cm-1
,
τv of 23 ( 2 ps, and the static ZFS of 0.024 ( 0.005 cm-1
.
Figure 4 shows that higher field data are in reasonably good
agreement with the best-fit profiles obtained from relaxation data
measured up to 40 MHz.
’ EXPERIMENTAL SECTION
The overall agreement between the calculated curves and the
experimental data shows that the main features of all profiles can
be reproduced as the result of an increase in τR upon conjugation
of 1 to 3. (Table 3) The change in the values of electron
relaxation parameters between 1 to 3 (similar to that observed
for other gadolinium complexes when bound to macro-
molecules)27,60,61 is likely determined by the simultaneous
presence of both static and transient ZFS, which is not fully
accounted for in fast rotating systems by available fitting
programs.53,62 Both profiles of 1 and 3 were fit according to
the “modified Florence” approach, and the main features of all
profiles could be reproduced as the result of an increase in τR on
passing from 1 to 3 (see Supporting Information, Figure S1).
Independent from the electronic parameters, the analysis clearly
shows that the NMRD profiles of 3 can only be reproduced for
tumbling times that are approximately 1 order of magnitude
larger than those of 1.
Conjugation of 1 to 3 results in an increase in τR from 0.049 to
0.60 ns at 310 K resulting in a 2-fold increase in relaxivity at low
field and a 5-fold increase at the peak value of 40 MHz. The
analysis of the data is consistent with an essentially unaltered
hydration of the Gd(III) complex upon conjugation to the
scaffold and shows that the increase of relaxivity is due to the
increase in τR. The 10-fold increase in τR upon the conjugation of
1 to 3 places the molecule in the intermediate τR range between
(0.5 and 4 ns). This range has been predicted to be optimal for
imaging at higher magnet field strengths.24
General Synthetic Methods. Unless noted, materials and sol-
vents were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO) and used without further purification. GdCl3 6H2O and 1,4,7,10-
3
tetraazacyclododecane (cyclen) were purchased from Strem Chemicals
(Newburyport, MA) and used without further purification. Unless
noted, all reactions were performed under a nitrogen atmosphere.
THF, acetonitrile, and dichloromethane were purified using a Glass
Contour Solvent system. Deionized water was obtained from a Millipore
Q-Guard System equipped with a quantum Ex cartridge (Billerica, MA).
Thin-layer chromatography (TLC) was performed on EMD 60F 254
silca gel plates. Visualization of compounds was accomplished using
either an iodoplatinate or UV light. Standard grade 60 Å 230-400 mesh
silca gel (Sorbent Technologies) was used for flash column chromato-
graphy.
1H and 13C NMR spectra were obtained on a Bruker 500 MHz
Avance III NMR Spectrometer or a Varian Inova 400 MHz NMR
spectrometer with deuterated solvent as noted. Electrospray ionization
mass spectrometry (ESI-MS) spectra were taken on a Varian 1200 L
single-quadrupole mass spectrometer. High resolution mass spectro-
metry data was aquired on an Agilent 6210 LC-TOF (ESI, APCI, APPI).
Analytical reverse-phase HPLC-MS was performed on a Varian Prostar
500 system with a Waters 4.6 ꢀ 250 mm 5 μM Atlantis C18 column.
This system is equipped with a Varian 380 LC ELSD system, a Varian
363 fluorescence detector, and a Varian 335 UV-vis detector. Pre-
parative runs were performed on a Water 19 ꢀ 250 mm Atlantis C18
Column. The mobile phases consisted of Millipore water (A) and
HPLC-grade acetonitrile (B). HPLC method 1: 0-5 min 100% A,
5-24:08 min 57.5% A, 24:08-30 min 0% A, 30-35 min 0% A, 35-40
min 100% A.
MR images were obtained of complexes 1 and 3 in glass
capillaries (1 mm) at 7 T (Figure 5). Molecular concentrations
ranged from 60 to 7.5 μM. The image was obtained using a RARE
pulse sequence with a TE and TR of 11 and 200 ms. Not
surprisingly, 3 is clearly brighter than 1 at the same molecular
Determination of r1 was accomplished using a Bruker minispec 60
MHz (1.41 T) magnet and a Varian Inova 400 MHz (9.4 T) NMR
spectrometer. At 1.41 T, the T1 relaxation times were determined using
an inversion recovery method, while at 9.4 T a saturation recovery
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Journal of the American Chemical Society
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method was used. The saturation recovery method utilized a 2 s
presaturation pulse centered on the water frequency. All measurements
were done at 37 °C at an approximate 1 mM concentration of CA in
10 mM DPBS purchased from Invitrogen.
protecting groups; once complete the formic acid was evaporated on a
rotary evaporator. The resulting oil was redissolved in water 3 ꢀ 10 mL
and evaporated to remove most of the formic acid. The resulting glassy
solid was dissolved in 30 mL of water and Gd(OAc)3 6H2O was added.
3
NMRD measurements were performed with a Stelar Spinmaster
FFC-2000-1T fast field cycling relaxometer in the 0.01-40 MHz proton
Larmor frequency range at 298 and 310 K. Standard field cycling
protocol was used. Longitudinal water proton relaxation rates were
obtained with an error smaller than 1%. Proton nuclear magnetic
relaxation dispersion (NMRD) profiles were obtained by plotting
proton relaxation rates as a function of applied magnetic field after
subtraction of the diamagnetic contribution of buffer alone and normal-
ization to 1 mM Gd(III) concentration.
1-Azido-3-chloropropan-2-ol (6) . 6 was synthesized as de-
scribed by Ingham et al with the following modifications.63 Diethyl ether
was used in the place of dichloromethane in the procedure. The final
product was not distilled as in the literature procedure but simply
extracted into ether and evaporated. The product was used directly in
subsequent reactions. Caution: Safe handling procedures for perchlo-
rates and small molecule azides should be reviewed before performing
this reaction, as there is a danger of explosion if heat, friction, or shock is
applied.
Tritert-butyl 2,20,200-(1,4,7,10-tetraazacyclododecane-1,
4,7-triyl)triacetate hydrobromide (tris-t-butyl-DO3A HBr).
Tris-t-butyl-DO3A HBr was synthesized according to the procedure
of Oskar with the following modifications.64 To a 500 mL RB flask was
added 10.279 g (59.8 mmol) of cyclen to which was added 14.8405 g
(179.2 mmol) of NaOAc. The solids were dissolved in 180 mL of
dimethylacetamide (DMA). The reaction was cooled to 0 °C with ice
and 26.5 mL (179.3 mmol) of tert-butyl bromoacetate dissolved in
70 mL of DMA was added dropwise over 40 min at 0 °C. The reaction
was allowed to warm to RT and stirred for two days and was poured into
a solution of 16.6 g of KBr in 1000 mL of H2O. The solution was brought
to a basic pH with 17.7 g (3.5 equiv) of NaHCO3. (Caution: A large
amount of gas is produced.) A total of 10 mL of ether was added to
initiate precipitation of the HBr salt of tris-t-butyl-DO3A. The final
white to off white powder was filtered and dried under a vacuum to give a
yield of 21.4976 g (60% yield). 1H NMR (500 MHz, CDCl3) δ 3.56-
2.57 (m, 21H), 1.46 (d, J = 3.6 Hz, 27H). 13C NMR (126 MHz, CDCl3)
δ 170.53, 169.63, 81.86, 81.71, 77.29, 77.04, 76.78, 58.22, 51.31, 51.12,
49.14, 47.53, 28.23, 28.19, 28.03, 0.00.
The pH was adjusted to ∼6.5 with 1 M NaOH and the reaction was
heated to 50 °C. The pH was adjusted back to 6.5 every 6-10 h until no
further change occurred (typically 1-2 days). The reaction was evaporated
and purified by reverse phase HPLC according to method 1, retention
time of 15.5 min, and 99.9% purity, followed by lyophilization to yield 412 mg
of 1 as a white powder in 41% yield based on the starting mass
of tris-t-butyl-DO3A. M/Z observed: 597.13151, calculated: 597.13367
[M þ H]þ.
Ethyl 5-(2,4,6-tribromophenoxy)pentanoate (8). To a
100 mL round-bottom flask was added 970.4 mg (2.933 mmol) of
tribromophenol, 483.1 mg (3.500 mmol) of K2CO3, and 0.5 mL (3.120
mmol) of ethyl 5-bromovalerate. The flask was charged with 30 mL of
dry DMF and nitrogen gas was bubbled through the reaction for 5 min.
The reaction was left under nitrogen and brought to 50 °C and stirred for
12 h. Once the reaction was shown to be complete by TLC (9:1 Hex:
EtOAc) the reaction was diluted with 50 mL of H2O and extracted three
times with 50 mL of diethyl ether. Afterward, the combined organic
layers were dried over MgSO4, filtered, and concentrated using a rotary
evaporator. The residue was purified by column chromatography (9:1
hexane: ethyl acetate) to give 1181.8 mg of a yellow to clear oil (88%
yield). 1H NMR (500 MHz, CDCl3) δ 7.62 (s, 2H), 4.12 (q, J = 7.1 Hz,
2H), 3.97 (t, J = 5.8 Hz, 2H), 2.40 (t, J = 4.6 Hz, 2H), 1.88 (dt, J = 6.5, 3.2
Hz, 4H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
173.70, 153.04, 135.21, 119.27, 117.49, 73.13, 60.54, 34.17, 29.55, 21.66,
14.48. M/Z observed: 456.8653, calculated: 456.8644 [MþH]þ.
Ethyl 5-(2,4,6-tris((trimethylsilyl)ethynyl)phenoxy)-
pentanoate (9). To a flame-dried 50 mL round-bottom flask was
added 0.5793 g (1.265 mmol) of S-8, 29.3 mg (0.154 mmol) of copper(I)
iodide, and 107.2 mg (0.4092 mmol) of triphenylphosphine. The flask was
charged with 12 mL of dry triethylamine. Nitrogen was bubbled through the
solution for 5 min followed by the addition of 1.8 mL (13 mmol) of TMS-
acetylene and 90.4 mg (0.128 mmol) of bis(triphenylphosphine) palladium-
(II) chloride were added. The flask was heated to 75 °C and left to stir
overnight under nitrogen. The reaction was concentrated using a rotary
evaporator. Thirty milliters of hexane was added to the flask and the
remaining solids were filtered off. The hexane solution was concentrated
using a rotary evaporator and the final residue was purified by silica gel
chromatography (40:1 hexane/ethyl acetate). 606.7 mg of clear oil was
obtained (93.8% yield). 1H NMR (500 MHz, CDCl3) δ 7.46 (s, 2H), 4.21
(t, J = 5.9 Hz, 2H), 4.10 (d, J = 7.1 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 1.93-
1.73 (m, 4H), 1.23 (t, J = 7.1 Hz, 3H), 0.27 - 0.13 (m, 27H). 13C NMR
(126 MHz, CDCl3) δ 173.66, 161.79, 137.59, 118.55, 117.80, 103.10,
100.05, 99.86, 94.69, 73.70, 60.46, 34.27, 29.96, 21.87, 14.46, 0.08, 0.01. M/Z
observed: 511.2505, calculated: 511.2515 [M þ H]þ.
Tritert-butyl 2,20,200-(10-(3-azido-2-hydroxypropyl)-1,4,7,
10-tetraazacyclododecane-1,4,7-triyl)triacetate (7). To a
250 mL round-bottom was added 4.9963 g (8.397 mmol) of tris-t-
butyl-DO3A and 2.8957 g (20.98 mmol) of K2CO3. The flask was
charged with 80 mL of anhydrous acetonitrile. The flask was sealed
and placed under a nitrogen atmosphere. 1.6957 g (12.50 mmol) of 6
was dissolved in 5 mL of acetonitrile and added to the solution of tris-
t-butyl-DO3A. The reaction was heated to 50 °C and stirred over-
night. The reaction was monitored by mass spectrometry to follow
the disappearance of the starting materials. 0.399 g (2.94 mmol) of 6
was added and allowed to stir for another 24 h. The reaction was
checked by MS to determine the disappearance of tris-t-butyl-DO3A.
Once all of the tris-t-butyl-DO3A had disappeared, the reaction was
filtered and evaporated to provide a yellow oil. The oil was dissolved
in minimal methanol and 100 mL of diethyl ether was added and the
flask was placed at -20 °C overnight. 3.968 g (77% yield) of clear to
yellow crystals of 7 formed overnight in the freezer and were filtered
and washed with cold ether. M/Z observed: 614.5, calculated: 614.4
[M þ H]þ.
Ethyl 5-(2,4,6-triethynylphenoxy)pentanoate (10). To a
100 mL RB flask was added 214 mg (0.418 mmol) of S-9 which was
dissolved in 20 mL of ethanol. To this was added 366 mg (6.29 mmol) of
KF and stirred for 4 h or until the reaction is complete by TLC (19:1
hexanes/ethyl acetate). Once complete the reaction was evaporated and
the solids were washed with hexanes. The crude product was purified by
column chromatography using 19:1 hexanes/ethyl acetate to provide
116 mg of a colorless to yellow oil in 94% yield. 1H NMR (499 MHz,
CDCl3) δ 7.53 (s, 2H), 4.23 (t, J = 5.9 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H),
3.26 (s, 2H), 3.01 (s, 1H), 2.37 (t, J = 7.2 Hz, 2H), 1.96 - 1.73 (m, 4H),
1.23 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 173.74, 162.57,
138.31, 117.76, 117.21, 82.72, 81.55, 78.72, 77.95, 74.05, 60.46, 34.18,
29.75, 21.69, 14.47, 14.40. M/Z observed: 295.1322, calculated:
295.1329 [M þ H]þ.
1-(3-Azido-2-hydroxypropyl)-4,7,10-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododecyl-gadolinium(III)(1). 1.0201 g
(1.661 mmol) (7) was dissolved in 100 mL of formic acid and
heated overnight. MS was used to observe the removal of the t-butyl
5-(2,4,6-Triethynylphenoxy)pentanoic acid (2). 105 mg
(0.375 mmol) of S-10 was dissolved in 5 mL of 1,4 dioxane. To this
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Journal of the American Chemical Society
ARTICLE
was added 1 mL of 1 M NaOH and the mixture was stirred for 4 h or until
completion. 9:1 hexanes/ethyl acetate was indicated by TLC. Five
milliliters of water was added to the reaction and the dioxane was
evaporated on a rotary evaporator. The remaining water was further
diluted by 5 mL of water and acidified with 3 M HCl. Upon reaching an
acidic pH, the product precipitated as yellow to orange crystals 86 mg,
91% yield. 1H NMR (499 MHz, CDCl3) δ 7.56 (s, 2H), 4.26 (t, J = 5.9
Hz, 2H), 3.29 (s, 1H), 3.04 (s, 1H), 2.48 (t, J = 7.3 Hz, 2H), 2.01-1.80
(m, 4H). 13C NMR (126 MHz, CDCl3) δ 178.25, 162.27, 138.12,
117.61, 116.99, 82.55, 81.30, 78.47, 77.78, 73.72, 33.37, 29.41, 21.24. M/
Z observed: 265.0865, calculated: 265.087 [M - H]-.
Excellence initiative at Northwestern University Award No.
U54CA119341.
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’ ASSOCIATED CONTENT
S
Supporting Information. The experimental section
b
(PDF) and X-ray crystallographic files (CIF) material are avail-
able free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
tmeade@northwestern.edu
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’ ACKNOWLEDGMENT
The authors would like to thank Charlotte Stern for crystal
structure analysis, Renee Strauch, Drs. Emily A. Waters, Ellen
Kohlmeir, and Keith MacRenaris for helpful discussions. A
portion of this work was completed at the Northwestern Uni-
versity Integrated Molecular Structure Education and Research
Center. Full funding disclosure can be found at http://pyrite.
chem.northwestern.edu/. We acknowledge the financial support
of the National Institute of Biomedical Imaging and Bioengineer-
ing Award No. 1R01 EB005866, NSF CHE-9871268, and the
National Cancer Institute Center for Cancer Nanotechnology
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