Analysis of the conformational behavior and stability of the SAP and TSAP isomers of lanthanide(III) NB-DOTA-type chelates

Article abstract of DOI:10.1021/ic2012843

Controlling the water exchange kinetics of macrocyclic Gd³⁺ chelates, a key parameter in the design of improved magnetic resonance imaging (MRI) contrast media, may be facilitated by selecting the coordination geometry of the chelate. The water exchange kinetics of the mono- capped twisted square antiprism (TSAP) being much closer to optimal than those of the mono capped square antiprism (SAP) render the TSAP isomer more desirable for high relaxivity applications. Two systems have been developed that allow for selection of the TSAP coordination geometry in 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (DOTA)-type Gd³⁺ chelates, both based upon the macrocycle nitrobenzyl cyclen. In this paper we report investigations into the stability and formation of these chelates. Particular focus is given to the production of two regioisomeric chelates during the chelation reaction. These regioisomers are distinguished by having the nitrobenzyl substituent either on a corner or on the side of the macrocyclic ring. The origin of these two regioisomers appears to stem from a conformation of the ligand in solution in which it is hypothesized that pendant arms lie both above and below the plane of the macrocycle. The conformational changes that then result during the formation of the intermediate H₂GdL⁺ chelate give rise to differing positions of the nitrobenzyl substituent depending upon from which face of the macrocycle the Ln³⁺ approaches the ligand.

Full text of DOI:10.1021/ic2012843

ARTICLE
pubs.acs.org/IC
Analysis of the Conformational Behavior and Stability of the SAP and
TSAP Isomers of Lanthanide(III) NB-DOTA-Type Chelates
†
‡
§
§
,‡,||
Gyula Tircso, Benjamin C. Webber, Benjamin E. Kucera, Victor G. Young, and Mark Woods*
†
Department of Inorganic and Analytical Chemistry, University of Debrecen, P.O. Box 21, Egyetem t ꢀe r 1, Debrecen H-4010, Hungary
‡
Department of Chemistry, Portland State University, 1719 SW 10th Avenue, Portland, Oregon 97201, United States
§
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, Minnesota 55455, United States
Advanced Imaging Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, L485, Portland,
Oregon 97239, United States
S Supporting Information
b
ABSTRACT: Controlling the water exchange kinetics of macro-
3
+
cyclic Gd chelates, a key parameter in the design of improved
magnetic resonance imaging (MRI) contrast media, may be
facilitated by selecting the coordination geometry of the chelate.
The water exchange kinetics of the mono- capped twisted square
antiprism (TSAP) being much closer to optimal than those of
the mono capped square antiprism (SAP) render the TSAP
isomer more desirable for high relaxivity applications. Two
systems have been developed that allow for selection of the
3+
TSAP coordination geometry in 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-type Gd chelates, both based
upon the macrocycle nitrobenzyl cyclen. In this paper we report investigations into the stability and formation of these chelates.
Particular focus is given to the production of two regioisomeric chelates during the chelation reaction. These regioisomers are
distinguished by having the nitrobenzyl substituent either on a corner or on the side of the macrocyclic ring. The origin of these two
regioisomers appears to stem from a conformation of the ligand in solution in which it is hypothesized that pendant arms lie both
above and below the plane of the macrocycle. The conformational changes that then result during the formation of the intermediate
+
H GdL chelate give rise to differing positions of the nitrobenzyl substituent depending upon from which face of the macrocycle the
2
3
+
Ln approaches the ligand.
’
INTRODUCTION
result of their small size. However, in targeted imaging ap-
proaches envisioned for future molecular imaging applications
the contrast agent must bind to a large biological target that
would slow this molecular rotation. Under these circumstances
suboptimal water exchange kinetics will limit relaxivity. It is
therefore not surprising then that considerable effort has been
Water exchange is one of several key parameters in the
function of magnetic resonance imaging (MRI) contrast agents.
The protons of water molecules coordinated to a paramagnetic
metal ion are characterized by extremely short longitudinal
relaxation time constants (T ). Exchange between these protons
and those of the bulk solvent causes the T of the bulk water to
become shorter. Clearly the length of time that a water molecule
resides upon the metal ion is crucial to this T -shortening effect;
too short and the protons do not have sufficient time to be relaxed,
too long and a relaxed water molecule needlessly occupies a
coordination site on the metal ion. So controlling the water
exchange rate of an MRI contrast agent, which generates increased
image contrast through the shortening of tissue T 's, is critical to
improving its function.
The Gd ion is the most commonly used paramagnetic ion
in MRI contrast media because of its high spin state and long
electron relaxation time. However, the water exchange kinetics
1
3+
directed to accelerating the water exchange kinetics of Gd
1
2ꢀ8
6,7
chelates.
Our own efforts in this area have been very
3+
successful affording Gd chelates that are optimal for max-
imizing relaxivity at 1.5 T, currently the most common mag-
netic field strength used for clinical imaging studies.
1
The rationale behind our approach was to modify the
solution state behavior of the popular MRI contrast agent
GdDOTA (Dotarem). In solution GdDOTA exists as a mix-
ture of two isomers, a monocapped square antiprism (SAP)
and a monocapped twisted square antiprism (TSAP). These
structures are in dynamic equilibrium in solution, intercon-
1
3
+
9
,10
verting by either ring flip or pendant arm rotation.
The
3
+
significance of this isomerism is that water exchange has been
of the Gd chelates in clinical use as contrast agents are much
slower than the optimal value determined by theory. This does
1
not limit the efficacy (termed relaxivity) of these agents because
their relaxivity is limited by rapid molecular reorientation, a
Received: June 14, 2011
Published: August 05, 2011
r 2011 American Chemical Society
7966
dx.doi.org/10.1021/ic2012843
|Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
3+
found to occur upward of an order of magnitude more rapidly
Chart 1. Formulae of Cyclen-Based Ligands for Ln Ions
1
1ꢀ13
in the TSAP isomer than in the SAP.
However, in solution
14
GdDOTA predominates as the SAP isomer (∼85% SAP).
3
+
Thus, constraining a Gd chelate to adopt solely a TSAP
isomer would be desirable for development of contrast agents
for targeted molecular imaging applications. Appropriately
substituting both the macrocyclic ring and the pendant arms
of a GdDOTA-type chelate, as in NB-DOTMA or 1 (Chart 1),
6
,7
can halt both these processes of isomer interconversion.
Furthermore, by ensuring that the configuration of the chiral
centers in both locations, ring and pendant arms, are the same
one may “select” a ligand that will afford a chelate that is
6
,7
exclusively TSAP, that is, S-SSS-1.
In addition to more rapid water exchange kinetics that
3
+
confer higher relaxivities upon Gd chelates used as targeted
MRI contrast agents, it is critical to also consider the stability of
these chelates. A targeted contrast agent is expected to reside in
the body much longer than a conventional agent; bound to its
3
+
target it must not allow the central, and toxic, Gd ion to be
released until after the agent has been excreted from the body.
3
+
The thermodynamic and kinetic stability of Ln chelates of S-
RRRR-NB-DOTMA and S-SSS-1, which will adopt exclusively
SAP and TSAP isomers, respectively, are therefore of con-
siderable interest. In this report we examine the thermody-
namic stability, as well as conformational and kinetic aspects of
the formation of these isomerically “selective” chelates.
’
EXPERIMENTAL SECTION
General Remarks. All solvents and reagents were purchased from
commercial sources and used as received. HPLC purifications were
performed on Water δ-Prep 150 HPLC system using a Phenomenex
Luna C-18 reversed-phase (50 ꢁ 250 mm) column. LC-MS analysis was
performed on a Thermo Scientific Accela pump with autosampler using
1
a Thermo Scientific LTQ XL/LTQ Orbitrap Discovery. H NMR
Crystallography. A crystal (approximate dimensions 0.20 ꢁ 0.10 ꢁ
3
spectra were recorded on a Varian Mercury, Bruker Avance IIa or
Avance III spectrometer operating at 300.01, 400.13, or 600.13 MHz,
respectively. C NMR spectra were recorded on a Varian Mercury
0.10 mm ) of H DOTMA(HCl) (H O) was placed onto the tip of a
4
2
2
3
0.1 mm diameter glass capillary and mounted on a Bruker SMART
13
16
Platform CCD diffractometer for a data collection at 173(2) K.
A
spectrometer operating 75.47 MHz. The syntheses of S-RRRR-NB-
DOTMA-tetraethyl ester and S-SSS-1 have been described previously.
preliminary set of cell constants was calculated from reflections har-
vested from three sets of 20 frames. These initial sets of frames were
oriented such that orthogonal wedges of reciprocal space were surveyed.
This produced initial orientation matrices determined from 58 reflec-
tions. The data collection was carried out using MoKR radiation
(graphite monochromator) with a frame time of 60 s and a detector
distance of 4.897 cm. A randomly oriented region of reciprocal space was
surveyed to the extent of one sphere and to a resolution of 0.84 Å. Four
major sections of frames were collected with 0.30° steps in ω at four
different ϕ settings and a detector position of ꢀ28° in 2θ. The intensity
7
A synthetic procedure for the preparation of DOTMA is provided in a
15
companion paper.
1R,4R,7R,10R)-R,R ,R ,R -Tetramethyl-[(S)-2-(p-nitro-
0
00 000
(
benzyl)]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tic Acid (S-RRRR-NB-DOTMA). To a solution of S-RRRR-NB-
DOTMA-tetraethyl ester (0.20 g, 0.28 mmol) in tetrahydrofuran
(5 mL) was added a 1.0 M solution of sodium hydroxide (1.13 mL).
Water (4 mL) was added to the reaction mixture which was then stirred
at 50 °C for 18 h. The solvents were removed under reduced pressure,
and the residue taken up into water (20 mL). The resulting solution was
filtered through a 0.45 μm syringe filter prior to purification by
preparative HPLC. Preparative HPLC purification was performed on
a C-18 column using water (0.037% HCl) as eluent for 5 min and then a
linear gradient to 40% MeCN and 60% water (0.037% HCl) after
17
data were corrected for absorption and decay (SADABS). Final cell
constants were calculated from the xyz centroids of 2488 strong
18
reflections from the actual data collection after integration (SAINT).
Please refer to Table 4 for additional crystal and refinement information.
1
9
The structure was solved using SHELXS-97 and refined using
1
9
SHELXL-97. The space group P2 2 2 was determined based on
1 1 1
ꢀ
1
3
5 min, at a flow rate of 50 mL min . Absorbance was monitored at 205
systematic absences and intensity statistics. A direct-methods solution
was calculated which provided most non-hydrogen atoms from the
E-map. Full-matrix least-squares/difference Fourier cycles were per-
formed which located the remaining non-hydrogen atoms. All non-
hydrogen atoms were refined with anisotropic displacement parameters.
The carboxylic acid and water protons were placed from the difference
map and were refined with relative isotropic displacement parameters.
The OꢀH bond distances for the water protons were placed at 0.84 Å
using the HIMP instruction. All remaining hydrogen atoms were placed
and 270 nm. After removing the solvents by freeze-drying, the title
compound was obtained as a colorless solid (0.07 g, 38%).
1
HPLC R
T
= 28.15 min; Mp = 193 °C, decomposes; H NMR (400
3
MHz, D O, pD 3), δ = 8.17 (2H, s br Ar), 7.43 (2H, d br, J = 8 Hz,
2
HH
3
Ar), 4.12ꢀ2.74 (21H, m br, J = 8 Hz, CH Ar, ring NCH , NCH),
HH
2
2
ꢀ
1
.63ꢀ1.26 (12H, m br, CH
Anal. Found C = 45.8%, H = 6.9%, N = 9.9%, C27
HCl) requires C = 46.0%, H = 6.7%, N = 9.9%.
3
); m/z (ESMS ESI-): 594 (100%, [H
3
L] ),
H
41
N
5
O
10 2(H
2
O) 2
3
3
(
7
967
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
2
2
in ideal positions and refined as riding atoms with relative isotropic
displacement parameters. The final full matrix least-squares refinement
converged to R1 = 0.0441 and wR2 = 0.1085 (F , all data).
Stability Determinations and Formation Kinetics. Preparation
PSEQUAD software was then used to process the titration data,
calculating the protonation and stability constants using eqs 1 and 2. For
2
3+
each Ln system the Irving factor was determined before and after the
pH measurement of the samples had occurred. The average of these
values was used to correct the measured pH readings for differences
caused primarily by the diffusion potential.
and Standardization of Stock Solutions. Stock solutions of lanthanide
3+
3+
3+
3+
chlorides (Ln = Ce , Gd , and Yb ) were prepared by dissolving the
corresponding Ln in concentrated hydrochloric acid. Excess HCl
2 3
O
½
HiLꢂ
KiH
¼
ð1Þ
ð2Þ
acid was removed by repeated evaporation to dryness until dissolution of
the residue in water afforded a solution with a pH in the range 4.5ꢀ5.5.
The concentrations of the stock solutions so prepared were determined
þ
½
Hiꢀ1Lꢂ½H ꢂ
by complexometric titration using a standardized Na
in the presence of xylenol orange as an indicator. The Na
2
H
2
EDTA solution
EDTA
½MLꢂ
KML ¼
2
H
2
½Mꢂ½Lꢂ
solution was standardized with ZnCl2 stock solution (prepared by
dissolving metallic zinc (99.9%, Merck) in diluted HCl solution) using
eriochrome black T as an indicator). Stock solutions of the ligands were
prepared from solid samples of the ligand by dissolution in water and
filtering through a 0.45 μM membrane filter. The concentration of the
ligand stock solutions were determined by potentiometric titration using
Kinetics of Formation. Because of the relatively high stability of the
+
intermediates H GdL obtained by direct pH-potentiometric titrations,
2
the chelate formation was studied for mixtures with 10-fold ligand excess
1.00 mM Gd and 10.0 mM ligand). Under these conditions the
3
+
(
saturation value of the formation rate constant is likely to be obtained
a standardized KOH (0.1817 M) solution kept under N , both in the
2
ꢀ1
ꢀ1
2+
2+
(kobs (s ) = k (s )). This was confirmed for acidic samples
r
absence and presence of a large excess of Ca ([Ca ]/[L] ratio was
approximately 40). Prior to titration the pH of each ligand solution was
set to about 1.75ꢀ1.80 by addition of a measured volume of strong acid
experimentally (for which the conditional stability is the lowest) by
varying the ligand concentration in the concentration range of 4.0 mM to
3.6 mM (pH 5.16 (S-RRRR-NB-DOTMA and S-SSS-1) and pH 4.37
1
(
HCl) of known concentration, facilitating measurements of the lowest
2+
and 4.67 (NB-DOTA). The pseudo-first-order rate constants (kobs)
protonation constants. In presence of Ca all the acidic protons
dissociate; thus the difference in base consumption in presence and
absence of Ca ion, which corresponds to two highly basic protons,
located on amines of the macrocycle. The protonation constants of each
ligand were calculated from the data obtained by titrating 1.8 and
obtained in the given concentration range were identical to the
uncertainty of the measurements (the reproducibility of kobs was found
to be in the range of 3ꢀ6%, as determined at low and high pH values for
2+
3
+
the GdNB-DOTA). The formation rates of the Gd chelates were
1
studied by following the changes in 1/T of the chelates over the course
2
.6 mM ligand samples with standardized KOH solution in the absence
2+
of the formation reaction. Longitudinal relaxation times were measured
using the “inversionꢀrecovery” pulse sequence (180°-τ-90°) using 6ꢀ8
different τ values on an MRS-6 NMR analyzer (Institut “Josef Stefan”,
Ljubljana, Slovenija), operating at 20 MHz. The sample temperature was
maintained at 25 °C with circulated air. Relaxometric data recorded as a
function of time were fitted to eq 3 to evaluate the first-order rate
constants with Scientist (Micromath) software using a standard least-
squares procedure.
of Ca over the pH range of 1.75ꢀ11.95.
Potentiometric Titrations. Potentimetric titrations were carried out
with a Thermo Orion EA940 expandable ion analyzer using Thermo
Orion semimicro combination electrode 8103BN in a thermostatted (at
2
5.0 °C) vessel. A Metrohm DOSIMATE 665 autoburet (5 mL
capacity) was used for base additions (of volumes not less than
.005 mL) and 1.0 M KCl was used to maintain the ionic strength.
0
The protonation constant determinations were carried out in 8.00 mL
sample volumes with magnetic stirring, and during the titrations, argon
ð ꢀ k tÞ
Rt ¼ Re þ ðR0 ꢀ ReÞ e
3
ð3Þ
3
gas was passed over the top of the samples to maintain a CO -free
2
environment. Standard buffers (Borax: 0.01 M, pH = 9.180 and KH-
phthalate 0.05 M, pH = 4.005) were used to calibrate the electrode. The
titrant, a carbonate-free KOH solution, was standardized against 0.05 M
KH-phthalate solution by pH-potentiometric method. The method
0 e t
where the R , R , and R are the longitudinal relaxation rates measured at
the start of the reaction (t = 0), at equilibrium condition, and at time t,
respectively)
Regioisomeric Dependence upon Reaction Conditions. A single
stereoisomer of S-RRRR-NB-DOTMA was obtained by preparative
20
+
proposed by Irving et al. was used to obtain H ion concentrations
from the measured pH values; therefore, “stoichiometric” constants
RP-HPLC. Stock solutions of ligand (56.4 mM) and YbCl
(170.3 mM) were prepared in water. A series of 1.0 M buffers
(KHSO /HOAc, pH 3.5), NaOAc/HOAc, pH 4.0, 4.5, 5.0), (MES,
pH 5.5, 6.0) were prepared, and the pH of each was adjusted at 60 °C.
Each buffer and stock solution was equilibrated at 60 °C prior to use.
Each buffer was used to prepare a sample 7.1 mM in ligand, 14.2 mM
3
were determined. The ionic product, pK of water was also calculated for
w
an acidꢀbase titration and was found to be 13.754.
4
3+
Owing to the slow rates of chelation involving Ln ions and 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-type ligands,
the stability constants of the chelates were determined using the “out-of-
cell” technique (also known as the batch method). Sixteen 1.5 mL
3
in YbCl , and with a total volume of 500 μL. Each sample was then
3+
samples of known ligand and Ln ion concentrations (approximately
sealed in a vial and incubated at 70 °C for 64 days. A second stock
2
mM each by applying slight 1.5ꢀ2% ligand excess) were prepared, and
solution of YbCl was prepared in 1.0 M NaOAc/HOAc buffer at pH
3
the total acid concentration in the samples was varied. The pH of the
samples was adjusted to a range within which chelation was expected to
take place, the pH range was approximated by equilibrium calculations
with the use of stability data published for LnNB-DOTA complexes.
Each sample was then sealed under a blanket of N and kept in an
5.0 (170.3 mM). This solution was used to prepare samples
of different concentration but at a fixed ligand/YbCl ratio (1:2) in
3
1.0 M NaOAc/HOAc buffer at pH 5.0. Solutions at [S-RRRR-NB-
DOTMA] = 0.5, 1.0, 2.5, 5.0, 10, 25, and 50 mM were prepared, also
sealed in a vial, and incubated for 64 days at 70 °C.
2
1
2
incubator at 40 °C for 2 months and then at 25 °C for a further 3 months.
The minimum time required to reach equilibrium was established by
preliminary relaxometric studies using the most acidic and basic samples
prepared for the Gd chelates in duplicates which were kept together
with the rest of the “out-of-cell” samples. When equilibrium had been
established the samples were opened and the equilibrium pH measured.
The samples were allowed to cool to room temperature for 24 h prior
to analysis. The ratio of regioisomeric products in each vial was
determined by LC-MS. Each sample was diluted to a concentration of
50 μM using 1:1 Methanol/Water. Each sample was then injected in a
250 ꢁ 2.0 mm Phenomenex luna C18(2) eluting with a linear gradient
from 100% water (0.05% formic acid) to 80% acetonitrile after 35 min,
3+
7
968
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
ꢀ1
with a flow rate of 375 μL min . The entire eluent flowed directly to an
ESI-MS detector monitoring for m/z = 765 in negative ionization mode.
The distribution of isomers was determined by manual integration of the
area of the peaks in the chromatogram. After LC-MS analysis all samples
1
were combined and purified by prep RP-HPLC. H NMR analysis of
each fraction was used to ensure that the peaks were assigned to the
correct regioisomer.
Epimerization Studies. A sample of YbS-SSS-1, purified by prepara-
tive RP-HPLC, was hydrogenꢀdeuterium exchanged by repeated D O
2
addition/freeze-drying cycles. After H/D exchange the sample was
2
dissolved in D O (pD1 DCl) (0.5 mL) such that the chelate concentra-
Figure 1. Schematic representations of the conformation of cyclen in
DOTA-type chelates. Left: A Dale’s representation of the [3333] ring
conformation of cyclen in the δδδδ conformation highlighting the differ-
ences between carbons located at a corner position versus those located at a
side position. Right: Highlighting a Newman projection of the ethylene
bridges, again in a δδδδ conformation, demonstrating that equatorial
positions on both carbons located at the corner and at the side are pro-S-.
tion was 20 mM. The sample was placed in a sealable 5 mm NMR tube,
1
and the tube sealed. A H NMR spectrum was recorded, and the sample
1
incubated in an oil bath at 90 °C. H NMR scans were acquired initially
at 1 week intervals, followed by 2ꢀ3 week intervals later in the study.
The ratio of regioisomers present in the study was determined by
integrating all three methyl resonances of each regioisomer. After
completion of the experiment, the sample was broken open, the solution
removed from the NMR tube, and a portion was analyzed by ESI-MS to
confirm the product mass (m/z = 751).
’
RESULTS AND DISCUSSION
Isomerism During Chelate Formation. We recently re-
3+
ported that the synthesis of Ln chelates of NB-DOTMA
ligands was complicated by the appearance of more than one
7
reaction product. Two principal reaction products were ob-
served and these had been attributed to racemization during the
alkylation of the amine in the 1-position of the macrocycle. Thus,
the two reaction products were attributed to the RRRR- and
SRRR- (SAP), and SSSS- and RSSS- (TSAP) isomers. It is now
clear that this rationalization is erroneous. The racemization
explanation was based primarily upon the observation that the
1
Figure 2. Expansion of the hyper-shifted region of the H NMR spectra
(
300 MHz, D O) of two isomeric chelates of YbNB-DOTMA (both
2
S
TSAP coordination isomers) focusing on the ax protons of the
macrocyclic ring. Previously these two isomers were ascribed to the S-
1
-position of the macrocycle is a much poorer nucleophile than
7
,24
SSSS- (top) and S-RSSS- (bottom) isomers. This assignment is now
understood to be erroneous, in fact these spectra correspond to two
regioisomers of the S-SSSS- isomer in which the nitrobenzyl substituent
is located on the corner (top) and on the side (bottom) of the
macrocyclic ring, see Figure 1.
the amines in other positions of the macrocycle. Indeed it is such
a poor nucleophile that even in the presence of 4 equiv of a triflate
as alkylating agent at room temperature it is possible to selec-
tively alkylate the other three amines of the macrocycle, leaving
7
the 1-position unreacted. This opened up the possibility of
introducing an achiral pendant arm in the 1-position eliminating
the perceived problem of racemization while maintaining the
structural integrity of the coordination geometry. In initial
experiments this approach appeared to be successful, affording
a wide frequency window allowing better resolution of the
chelate resonances. However, such an approach overlooks one
key aspect of the formation of chelates with nitrobenzyl sub-
stituted DOTA-type ligands: an aspect that never became
>
90% of a single isomeric chelate.
The synthesis of the tetra-ester of S-SSSS-NB-DOTMA
2
1
apparent during studies on the parent NB-DOTA chelates.
3+
afforded two compounds, present in roughly equal amounts,
In all known stable Ln chelates of DOTA-type ligands the
1
23
which could be distinguished by H NMR after the alkylation
macrocyclic ring adopts a [3333] ring conformation. This
reaction. Separation of these two compounds proved an intract-
able problem, and mass spectrometry showed that they had the
same mass. The ligand was taken through to the corresponding
places each amine on one of the side of a “square” ring and gives
rise to two distinct carbon environments: a carbon may be
either on the side or on the corner (Figure 1). Introducing a
single substituent onto this ring, the ring will orient itself so as
to place the substituent into an equatorial position, thus
minimizing torsional strain. In the case of NB-cyclen the
configuration of the chiral carbon is S-, and a δδδδ ring
conformation will place the substituent into an equatorial
position. This is the basis upon which the ring conformation
is locked and the coordination geometry selected. How-
ever, what was not considered in detail previously is that for
cyclen in a δδδδ conformation the equatorial protons on both
the carbons on the corner and the side are pro-S. This means
that a substituent can occupy one of two chemically distinct
positions while adopting an equatorial position and locking the
3+
Eu chelate, and two chelates, both in the expected TSAP
1
geometry, were observed by H NMR and HPLC. The assign-
ment to two diastereoisomers (differing in their configuration in
the 1-position) seemed a reasonable one, particularly once a
similar observation had been made for the chelates, but not the
esters, of S-RRRR-NB-DOTMA. This reliance upon assessing the
3
+
23
6,7
Ln chelate rather than the ligand proves to have been the major
error in these studies. There is a definite advantage to studying
3
+
1
chelates of Ln ions that induced large hyperfine shifts; the H
NMR of asymmetric ligands such as those studied here overlap
with one another making structural determinations very difficult,
3+
introducing a shifting Ln ion spreads these resonances out over
7
969
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
a
Table 1. Protonation Constants of the Ligands (25 °C)
b
c
S-RRRR-NB-DOTMA
S-SSS-1
DOTA
NB-DOTA
1.0 M KCl
NB-DOTA
I =
1.0 M KCl
11.81 (0.01)
8.23 (0.03)
4.00 (0.04)
3.46 (0.03)
2.13 (0.03)
1.0 M KCl
11.30 (0.01)
9.96 (0.01)
4.60 (0.05)
5.41 (0.01)
1.99 (0.01)
1.0 M KCl
12.09
9.68
0.1 M Me
10.93
9.14
4
NCl
log K
log K
log K
log K
log K
log K
1
2
3
4
5
6
10.00 (0.01)
9.54 (0.01)
4.29 (0.03)
4.72 (0.01)
2.28 (0.02)
H
H
H
H
H
4.55
4.50
4.13
4.19
2.33
1.40
H
∑
log K
i
29.62
33.26
30.45
30.83
32.49
ꢀ
3
ꢀ3
ꢀ3
fit
9.79 ꢁ 10 mL
425
5.00 ꢁ 10 mL
307
2.59 ꢁ 10 mL
128
c
data points
a
b
Values in bold highlight instances were the fourth pK
a
is higher than the third. Data taken from ref 27. Data taken from ref 21.
ring into a δδδδ conformation (Figure 1). Thus, for any singly
substituted cyclen there are two possible regioisomeric struc-
conformational change in the ligand. In this new conformation
the site of the fourth protonation is rendered more basic than
that same site in the initial conformation. If this change in
basicity is large enough then the next protonation step, in this
case the fourth one, may have a protonation constant that is
higher than the previous one. One might expect that such
situations are more likely to occur in highly rigid structures
such as S-SSS-1 and DOTMA in which conformational ex-
change processes are likely to be restricted. In the case of S-
SSS-1 this observation lead us to consider the possibility that
one of the two conformations places the nitrobenzyl substi-
tuent on the side of the macrocyclic ring, and the other places it
on the corner (Figure 1). At first glance it may also seem that
this would account for the observation of regioisomeric
2
4
tures, which may be identified from COSY NMR data.
3+
In the case of NB-DOTA, chelates of all the Ln ions were
1
prepared and analyzed by H NMR. In each case only two
structurally distinct chelates were identified corresponding to
21
one SAP (Λ(δδδδ) isomer and one TSAP (Δ(δδδδ) isomer.
These two isomers were found to interconvert by arm rotation
and in all cases where 2D-COSY data were acquired the
nitrobenzyl substitutent was found to lie on a carbon at a
2
1
corner. No sign of additional structures relating to regioisomer-
ism of the nitrobenzyl substituent were observed in any of the
NB-DOTA chelates. The first indication of regioisomerism arose
during a study of the NIR-CD properties of YbNB-DOTMA
2
4
1
3+
chelates. As part of this study 2D- H NMR experiments were
run on the chelates including a sample that had initially been
assigned as the S-SRRR- isomer (Figure 2). This study placed the
nitrobenzyl substituent on a carbon on the corner for the two
chelates for which studies were completed (one SAP and one
TSAP), but for the “S-SRRR-” isomer the nitrobenzyl substituent
was found to lie on a carbon on the side of the ring. Appropriate
weight was not given to the implication of this observation at the
time. It was not until experiments looking at the stability of the
chelates and the relative stability of each isomer were undertaken
that the significance of this observation became clear.
products when Ln chelates of S-SSS-1 are prepared. How-
ever, such an explanation does not really provide a satisfactory
explanation to this question, nor does it account for conforma-
tional changes in DOTMA that also exhibits a fourth pK that is
a
1
5
higher than its third.
As part of this study the pK values of NB-DOTA were
a
measured again, this time using 1.0 M KCl as the ionic back-
ground. The reasons for choosing KCl as the ionic background
2
8
have been expressed elsewhere, and while it offers certain
+
advantages it must be borne in mind that the K ion is not inert
in these systems. Comparing the protonation data obtained for
3+
Protonation Constants. The suitability of a Gd chelate for
in vivo application is in large part determined by its thermo-
dynamic stability. This may conveniently be achieved by means
NB-DOTA in KCl with that obtained in the more inert Me NCl
4
used initially, presents a more likely explanation for why some
fourth pK 's are higher than the third. The fourth pK of NB-
a
a
25
of potentiometric titration. The first stage of this process being
to determine the protonation constants of the ligand, informa-
tion which may also shine light into other aspects of the ligand’s
behavior. The protonation constants of S-RRRR-NB-DOTMA
and S-SSS-1 were determined by potentiometric titration using
DOTA in KCl is also higher than the third, but this is not the
+
case with Me NCl. It is known that the K ion interacts,
4
although weakly, with these types of amino carboxylate
2
9
+
ligands. Thus, it seems likely that when K is present in the
system it interacts with the carboxylates of the ligand during the
first two protonation steps which correspond to protonation of
the macrocycle. This interaction makes it more difficult to
1
.0 M KCl as an ionic background. Each ligand exhibited five
protonation constants, cf. the parent ligand DOTA (Table 1).
However, an unusual phenomenon was observed in the case of S-
protonate the carboxylates, thereby lowering the third pK . But
addition of that third proton displaces K from the ligand
altogether which renders the fourth protonation step more
favorable, in certain cases more favorable than the third. Such a
a
15
+
SSS-1. Like the protonation constants for DOTMA, the fourth
protonation constant of S-SSS-1 is actually higher than that of the
third. This phenomenon has been noted previously in other aza-
26
crown systems such as cyclam. In contrast, the third and fourth
scenario is impossible with Me NCl which cannot interact with
4
protonation constants of S-RRRR-NB-DOTMA follow the more
normal incremental ordering also observed for DOTA.
Although noted in other ligand systems, it is highly unusual
for a protonation constant to be higher than the one that
precedes it. One explanation for this unusal phenomenon is
that one protonation step, in this case the third one, leads to a
the ligand and so in this case the pK 's are observed to increase
a
27
incrementally. Thus, an alternative explanation for the produc-
tion of multiple regioisomeric chelates must be sought.
Chelate Formation. To probe why specific chelates are
formed it is first necessary to understand the mechanism by
3
+
which the chelates form. For Ln chelates of DOTA this
7
970
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
Table 2. Stability Constants of the Intermediate Chelates
+
H LnL for a Variety of DOTA-Type Ligands (I = 1.0 M
2
KCl, 25 °C)
log KH2LnL
+
a
b
c
d
EuDOTA
4.32; 4.40; 5.88; 8.26
e
GdDOTA
9.32
f
TbDOTA
8.96
GdNB-DOTA
GdS-SSS-1
5.43 (0.03)
6.06 (0.03)
4.70 (0.02)
GdS-RRRR-NB-DOTMA
a
I = 1.0 M NaCl, determined by potentiometric titration under non-
b
equilibrium conditions, taken from ref 30. I = 1.0 M NaCl, determined
c
by UVꢀvis spectrophotometry, taken from ref 30. I = 0.1 M KCl,
+
d
Figure 3. Mole fraction of H
pH for S-RRRR-NB-DOTMA (blue), DOTMA (black), NB-DOTA
red), and S-SSS-1 (green).
2
GdL present in solution as a function of
determined by luminescence, taken from ref 32. I = 0.1 M Me NCl,
4
determined by potentiometric titration under nonequilibrium condi-
e
(
tions, taken from ref 31. I = 0.1 M Me
4
NCl, determined by potentio-
f
metric titration, taken from ref 31. I = 0.1 M Me
potentiometric titration, taken from ref 31.
4
NCl, determined by
+
understand how the H GdL chelate could undergo rearrange-
2
ment so as to alter the position of a conformationally locked
macrocyclic ring. Even though the mechanism by which the two
trans- macrocyclic protons are removed remains the subject of
some conjecture, neither proposed mechanism would facilitate
the repositioning of the nitrobenzyl substituent. It seems more
3
0
mechanism was elucidated by Br €u cher and co-workers. In
3
+
30
this model the Ln ion approaches the DOTA ligand and
rapidly forms a weak intermediate chelate. Formation of this
intermediate chelate results in the rapid dissociation of pro-
tons from all the ligand carboxylates and displacement of at
least four water molecules from the inner hydration sphere of
the metal ion. This intermediate chelate is primarily electro-
static in nature. The metal ion is bound through interactions
with all four negatively charged carboxylate groups, and the
macrocyclic ring remains diprotonated (the first and second
pK of DOTA correspond to trans-protonation of the macro-
cycle), such that this intermediate has been identified to be of
the type H LnL . These two protons on the macrocyclic amines
must then dissociate from the intermediate H LnL chelate in
probable that the conformation of the ligand in solution and the
3+
direction of approach of the Ln ion during the rapid first
reaction step are the determining factors in the regiochemistry of
the final chelate.
Furthermore, the data in Table 1 and 2 allow the mole fraction
3+
of Gd to be calculated as a function of pH (Figure 3) for each
ligand. This is important from the point of view of determination
of the kinetics of formation of the fully formed chelates. Since the
a
+
ꢀ
rate determining step is the transformation H GdL f GdL +
2
+
30
+
2
2H , if accurate kinetic data are to be obtained it is critical that all
experiments be performed under conditions that ensure H GdL
+
+
2
2
3
+
the rate determining step before the Ln ion is able to drop
down into the coordination cage of the ligand. The nature of the
H LnL intermediate has recently been confirmed by potentio-
is the sole species in solution in the initial stages of reaction
+
(formation of H GdL is so rapid as to be considered instanta-
2
+
2
neous for the purposes of these measurements). From Figure 3 it
metric, luminescence, and EXAFS methods, although it has also
has been shown that over time a second intermediate complex
may form in which fewer water molecules are bound to the
is clearly seen that above ∼pH 4.2 all the ligands under
+
investigation herein are exclusively in the form H GdL . Accord-
2
ingly the kinetics measurements were made by measuring the
3
+
31
3+
Ln ion (q = 3).
Potentiometric titration of a ligand in the presence of Gd
decrease in Gd relaxivity upon complex formation at different
3+
pH values in the range 5.16ꢀ7.12. Reactions were run in the
allows the formation and in particular the stability of this
intermediate chelate to be probed. Because the formation of
the fully formed chelate is very slow, requiring the use of out-of-
cell potentiometric techniques for measurement vide supra, these
data are uncontaminated by subsequent formation of the fully
formed chelate. The potentiometric titrations of S-RRRR-NB-
DOTMA, S-SSS-1 as well as NB-DOTA in the presence and
presence of 5ꢀ14-fold excesses of ligand such that reactions were
pseudo-first-order and described by eq 4, where [Gd] is the total
t
3+
concentration of Gd and kobs the pseudo-first-order rate
constant.
d½GdLꢂ
¼
kobs½Gdꢂt
ð4Þ
dt
3
+
absence of Gd (Supporting Information) reveal that all
+
three ligands form the same H GdL type intermediate chelate
2
The relationship between pseudo-first-order rate constants
3+
on the way to the fully formed Gd chelate. These titration data
enable the stability constants of these intermediate chelates to be
kobs and ligand concentration is expressed by eq 5; in which k is
f
+
the rate constant characterizing the rearrangement of H GdL
2
ꢀ
+
C
determined (Table 2). No notable trend is immediately apparent
into GdL + 2H and the K
is the conditional stability
H2L
+
+
from these obtained values. However, the formation of H GdL
2
constant of the H GdL .
2
intermediates for all the ligands under study here (and also for
1
5
C
kf K
þ
½Lꢂ
H2GdL
DOTMA) is an important observation from the point of view
of the regiochemistry of the final chelates of S-RRRR-NB-
DOTMA and S-SSS-1. It strongly indicates that the regiochem-
istry of the final chelate is determined in this fast initial step of the
chelation mechanism since it is difficult, if not impossible, to
H GdL
C
2
kobs ¼ 1
ð5Þ
þ K
þ
½Lꢂ
Under conditions involving higher ligand concentrations, such as
C
those employed here, K H2L[L] . 1, and thus kobs ≈ k . In other
f
7
971
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
3
+
+
ꢀ
Table 3. Summary of the Kinetics of Formation of Gd
Chelates (I = 1.0 M KCl, 25 °C)
H GdL into GdL is negligible and the rate of chelate formation
2
ꢀ
is essentially determined by kOH (Table 3).
When the values of kOH
RRRR-NB-DOTMA are compared with those obtained for the
more flexible ligand systems GdNB-DOTA and EuDOTA it is
apparent that chelates of more substituted and therefore more
rigid ligand systems form more slowly than those of more flexible
ligand systems. It is noteworthy that NB-DOTA, which has a
more rigid macrocyclic ring system than DOTA, forms chelates
only marginally more slowly than DOTA but DOTMA, in which
the rigidity of the ligand system is introduced into the pendant
ꢀ
determined for GdS-SSS-1 and GdS-
ꢀ1
ꢀ1 ꢀ1
kH2O (s
)
OH
k ꢀ
(M
s )
3
3
a
6
DOTA
not detected
7.5 ꢁ 10
ꢀ
5
4
6
3
4
NB-DOTA
(9.1 ( 1.1) ꢁ 10
(1.4 ( 0.2) ꢁ 10
(ꢀ5.0 ( 0.8) ꢁ 10
(1.6 ( 0.8) ꢁ 10
(6.8 ( 0.4) ꢁ 10
(6.4 ( 0.3) ꢁ 10
ꢀ
S-SSS-1
ꢀ
5
S-RRRR-NB-DOTMA
a
Data published in ref 33 was re-examined according to eq 6.
3+
arms, exhibits a value of kOH (for the Eu ion) that is 3 orders of
ꢀ
33
magnitude smaller than DOTA. Indeed, all three of the chelates
substituted in the R-position of the pendant arms exhibit
substantially slower kinetics of formation. One may conclude
therefore that a significant change in pendant arm conformation
is required to allow the Ln ion to drop down into the
coordination cage and that R-substitution hinders these con-
formational changes.
3+
The values of k
and kOH determined here for S-SSS-1 and
ꢀ
H O
2
S-RRRR-NB-DOTMA are a weighted average of the values for
both regioisomers of each chelate (side and corner). Other than
identifying the key intermediate(s), the formation of which
seems to give rise to different regioisomers, these data afford
little information that can illuminate the question of why two
regioisomeric chelates are produced for these chelates. To
further probe this question a sample of H S-RRRR-DOTMA
6
was prepared and purified to a single isomer by prep-HPLC.
This sample was then used to probe the effect of pH and
concentration upon the regioisomeric ratio produced by pre-
3
+
paring the corresponding Yb chelate. 7.1 mM S-RRRR-
DOTMA were prepared in 1 M buffer solutions from pH 3 to
6
, 2 equiv of YbCl were then added. Additional samples were
3
prepared in 1.0 M NaOAc buffer at concentrations from 0.5 to
0 mM in ligand, again with 2 equiv of YbCl . All solutions were
5
3
sealed in vials and incubated for 64 days at 70 °C, and the
proportion of each regioisomer produced in each reaction was
determined by LC-MS monitoring at m/z = 765. Figure 4 shows
that in all cases two chelates are produced; the ratio of these
chelates is notable for not only its pH dependence but also its
concentration dependence.
The production of two isomeric chelates from a single
stereoisomer of ligand unambiguously demonstrates that our
earlier hypothesis (that the two chelates were the product of
racemization during the alkylation reaction) was erroneous.
The two chelates must instead differentiate themselves during
Figure 4. Ratio of YbS-RRRR-DOTMA chelates with the nitrobenzyl
substituent positioned on the corner of the macrocycle (YbNB-DOT-
MA (corner)) and that with the nitrobenzyl substituent positioned on
the side of the macrocycle (YbNB-DOTMA (side)) formed during
chelate formation (a) at 7.1 mM as a function of pH in a series of 1.0 M
buffers, (b) as a function of initial ligand concentration at pH 5.5 in 1.0 M
NaOAc buffer. All reactions were incubated for 64 days at 70 °C.
chelate formation, and almost certainly during the initial
+
words k dominates the value of the pseudo-first-order rate
formation of the H LnL intermediate chelate, vide infra. A
f
2
+
constant, kobs, when formation of the intermediate, H GdL , in
sudden break in the regioisomerism of the chelates as a function
of pH is not observed, consistent with the idea that pH
dependent conformational changes are not responsible for
the production of different regioisomers. The more gradual
changes observed for the regioisomeric ratio with changing pH
is suggestive of subtle changes in the relative rates of formation
(and possibly also the reverse reactions) of each regiosiomer
with changing pH. However, care should be taken not to draw
more detailed conclusions, even in a qualitative sense, from this
data. The model of all equilibria involved in these processes is
extremely complex, with many more quantities unknown than
are known. It is unclear which if any samples have reached
equilibrium. The choice of ligand/metal ratio was arbitrary and+
not designed to afford quantitative kinetic data. The use of Na
2
the initial stages of the reaction is complete. k maybe further
f
refined into the rate constants for water and base assisted
+
deprotonation of the H GdL , k
and kOH
H O
2
ꢀ
, respectively,
2
according to eq 6.
kf ¼ kH O þ kOHꢀ ½OHꢀꢂ
ð6Þ
2
Accordingly the experimentally determined values of the
pseudo-first-order rate constant, kobs (that approximates to kf)
were plotted against the concentration of hydroxide to afford
values of k
the value of k
and kOH
ꢀ
(Supporting Information). In each case
H O
2
was found to be small (Table 3) indicating that
H O
2
the contribution of water to deprotonation and rearrangement of
7
972
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
Table 4. Crystallographic Data for H DOTMA 2HCl 3H O
4
3
3
2
empirical formula
20 2 4 11
C H44 Cl N O
formula weight
587.49
173(2)
0.71073
temperature (K)
wavelength (Å)
crystal system
orthorhombic
P2
space group
1 1 1
2 2
a (Å)
b (Å)
c (Å)
R
9.5934(7)
15.8093(12)
18.7282(14)
90°
β
90°
γ
90°
2840.4(4)
4
3
volume (Å )
Z
density (calculated)
1.374
3
(Mg/m )
absorption coefficient
0.289
1256
ꢀ
1
(mm
)
F(000)
crystal color,
morphology
colorless, block
3
crystal size (mm )
θ range for data
collection
0.20 ꢁ 0.10 ꢁ 0.10
1.69 to 25.05°
index ranges
ꢀ11 e h e 11, ꢀ18 e k e 18,
22 e l e 22
ꢀ
reflections collected
24064
Figure 5. 50% Thermal ellipsoid plot of the crystal structure of
H
4
DOTMA 2HCl 3H
2
O. All hydrogen atoms, except those protonat-
independent reflections
observed reflections
5022 [R(int) = 0.0545]
4221
3
3
ing amines, have been omitted for clarity as have water molecules and
chloride counterions. All carboxylates are protonated on O2, O4, O6,
and O8.
completeness to θ = 25.05°
absorption correction
max. and min transmission
refinement method
99.9%
multiscan
0.9717 and 0.9445
full-matrix least-squares on F
5022/0/342
ꢀ
and AcO in several buffers means that labile buffer compo-
2
nents were employed in concentrations that are very high
relative to the reactants. Nonetheless, these experiments also
prove how it might have been possible to mistakenly believe
that substitution of an achiral arm into the 1-position had solved
data/restraints/parameters
2
goodness-of-fit on F
1.011
final R indices [I > 2σ(I)]
R indices (all data)
R1 = 0.0441, wR2 = 0.0976
R1 = 0.0607, wR2 = 0.1085
ꢀ0.13(8)
7
the problem of multiple isomers. In that case the chelation
absolute structure parameter
largest diff. peak and hole
reaction was performed at pH 5.5 at 210 mM. From the data in
Figure 4b it is clear that as the concentration of reactants is
increased, so increases the proportion of chelate with the
substituent on the corner. Although this increase is modest at
low concentrations, from 0.5 to 1 mM, as the concentration
ꢀ
3
0.872 and ꢀ0.476 e Å
crystals of H
DOTMA 2HCl 3H
O were grown from ∼5 M
4
3
3
2
HCl by slow evaporation at ∼40 °C. The structure obtained from
these crystals (Figure 5 and Table 4) shows the preorganized
nature of this ligand system that confers extremely high thermo-
dynamic stability upon its Ln chelates. Although they crystal-
lize in different space groups the structure of H DOTMA 2HCl
ꢀ
2
increases beyond 10 M this increase becomes much more
rapid. Extrapolating to 210 mM it is very possible that only
minimal quantities of a regioisomer with the substituent on the
side would have been produced in that reaction.
3+
25
4
3
Conformational Analysis. The ligand DOTMA represents a
convenient starting point for an analysis of the conformational
effects of regioisomerism of the nitrobenzyl substituent. In our
hands nitrobenzyl substituted chelates, such as those of NB-
DOTA, NB-DOTMA, and 1, do not crystallize but crash out of
solution as amorphous solids. In contrast DOTMA crystallizes
readily both as its hydrochloride or alkali metal salt. However,
since chelation reactions are necessarily run under acidic condi-
(P2 2 2 ) is highly reminiscent of that determined for
1 1 1
11
H DOTA 2HCl (P2 /n) (Figure 6). In each structure the
4
3
1
23
aza-crown adopts a [3333] conformation that confers the same
helicity upon each ethylene bridge (Table 5). This [3333] ring
conformation is notably more distorted in the structure of
H DOTMA, which is diagonally elongated, than it is in
6
H DOTA rendering each ethylene bridge in H DOTMA en-
6
6
tirely nonequivalent to all the others. This conformation places
all four pendant arms in each ligand structure on the same side of
the macrocycle. In each structure the pendant arms are twisted
into a slight helix that leads each ligand to resemble the structure
3
+
tions (Ln ions are poorly soluble at higher pH), it is the
protonated form of DOTMA that is more applicable as the
starting point of studies into ligand conformation. X-ray quality
7
973
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
Table 5. Selected Distance and Angles from the Crystal
Structures of H DOTMA 2HCl 3H O and
4
3
1
3
2
1
H DOTA 2HCl 5H O
4
3
3
2
parameter
H
6
DOTMA
parameter
6
H DOTA
Angles [deg]
ꢀ49.05
ꢀ69.84
ꢀ49.47
ꢀ68.83
10.41
N1ꢀCꢀCꢀN2
N2ꢀCꢀCꢀN3
N3ꢀCꢀCꢀN4
N4ꢀCꢀCꢀN1
N1ꢀCꢀCꢀO2
N2ꢀCꢀCꢀO3
N3ꢀCꢀCꢀO5
N4ꢀCꢀCꢀO7
N1ꢀN2ꢀN3
N2ꢀN3ꢀN4
N3ꢀN4ꢀN1
N4ꢀN1ꢀN2
O2ꢀO3ꢀO5
O3ꢀO5ꢀO7
O5ꢀO7ꢀO2
O7ꢀO2ꢀO3
N1ꢀCꢀCꢀN2
N2ꢀCꢀCꢀN3
N3ꢀCꢀCꢀN4
N4ꢀCꢀCꢀN1
N1ꢀCꢀCꢀO4
N2ꢀCꢀCꢀO1
N3ꢀCꢀCꢀO6
N4ꢀCꢀCꢀO7
N1ꢀN2ꢀN3
N2ꢀN3ꢀN4
N3ꢀN4ꢀN1
N4ꢀN1ꢀN2
O1ꢀO6ꢀO7
O6ꢀO7ꢀO4
O7ꢀO4ꢀO1
O4ꢀO1ꢀO6
ꢀ53.57
ꢀ59.76
ꢀ57.09
ꢀ60.39
4.19
ꢀ1.53
23.29
6.42
7.35
21.20
10.79
86.17
94.69
85.71
93.43
82.26
93.71
84.98
98.92
80.41
99.62
79.90
99.87
131.74
51.58
112.50
64.04
Distances [Å]
2.384
N ꢀO
4
4
2.507
The calculated energies of each resulting conformer are sum-
marized (Table 6). From these data it is clear that in this highly
protonated form, a ligand that accommodates the benzyl sub-
stituent on any corner of the macrocycle is lower in energy than
one that positions it on the side. Because each ethylene bridge is
rendered inequivalent by ligand distortion, each conformer with
the substituent on a corner has a different energy. However,
positioning the benzyl group on a corner with a larger dihedral
angle, C3 or C7 rather than C1 or C5, results in the lowest energy
conformations of all. Although this corresponds to the regioi-
somer that predominates under more concentrated reaction
conditions, these results cannot account for the production of
the second regioisomer in which the substituent is located on
the side.
Suitable crystals of a diprotonated form of DOTMA could not
be grown. An initial probe of the conformational preferences of
this protonation state was undertaken, again using the crystal
Figure 6. 50% Thermal ellipsoid plot of the crystal structure of
4 2
H DOTA 2HCl 5H O, reported in reference 11. All hydrogen atoms,
3
3
except those protonating amines, have been omitted for clarity as have
water molecules and chloride counterions. All carboxylates are proto-
nated; protons are located on O2, O3, O5, and O8.
observed in TSAP chelates rather more than that observed in
SAP chelates. Curiously, and despite that fact that LnDOTMA
15
chelates exhibit a preference for the TSAP isomer, the
7
NꢀCꢀCꢀO torsion angles of H DOTMA are greater than
6
those observed in the H DOTA structure, and this leads to a
6
shortening of the distance between the N and O planes relative
4
4
3+
to DOTA, the opposite situation to that observed in the Gd
1
5
chelates.
The conformations of both H DOTMA and H DOTA ob-
6
6
served in the crystal are reminiscent of how any DOTA-type
structure of H DOTMA as a starting point. The same MM+
6
34
ligand is commonly drawn in the mechanism of chelate
modeling procedure was used as before; however, on this
occasion all protons were removed from the carboxylate groups
30
formation elucidated by Br €u cher and co-workers. As such they
2
ꢀ
represent a logical starting point for an investigation into how the
to afford an H DOTMA species, and no part of the ligand
2
+
ligand conformation during the initial formation of H LnL
system was constrained during energy minimization. The results
2
chelates might affect the regioisomeric ratio of chelates. If this
conformation of ligand is predominant in solution then only if
both side and corner positions of the macrocycle can accom-
modate the nitrobenzyl substituent is it possible for both
regioisomeric chelates to be formed. A conformational analysis
was performed using MM+ molecular modeling calculations to
establish which of the eight equatorial positions of the aza-crown
of energy minimization afforded results very similar to those
obtained for H DOTMA; the lowest energy ligand conformation
6
was consistently obtained when the benzylic substituent was
positioned on the corner of the macrocycle. Notably the macro-
cyclic distortion present in the crystal of H DOTMA was
6
observed to reduce considerably during this procedure which
may account for the much lower energy gap observed for C1 in
this case. This seems to indicate that regardless of the proton-
ation state of the ligand it is not possible that all four pendant
arms adopt positions on the same side for the macrocyclic ring of
NB-DOTMA or 1. However, it may be that this is the case for
NB-DOTA which affords only a single regioisomer of SAP and
in H DOTMA accommodated a benzyl substituent with the
6
lowest energy penalty. A benzylic substituent was introduced
into each equatorial position in turn and an energy minimization
of the substituent performed while the structure of the remainder
of the ligand (macrocycle and pendant arms) was constrained.
7
974
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
Table 6. Calculated Energy Differences (MM+) of Ligand
Conformations Resulting from the Introduction of a Benzyl
same, and δ to accommodate the S- configuration at carbon.
However, to achieve this, and keep the two pendant arms that are
3+
Substituent into the Crystal Structure of H DOTMA and the
already interacting with the Ln ion in the same position, a
positional shift occurs. Every atom in the macrocycle shifts round
the ring one position. In this way the λ ethylene bridges can flip to
δ inducing a “flip” in the pendant arms such that all four are
6
a
Lowest Energy Conformation so Obtained
ΔE (kcal/Å mol)
3
+
positioned on the same side of the macrocyclic ring as the Ln
carbon
position
H
6
DOTMA
H
2
DOTMA
ion. Of course the consequence of this, which can only be
observed in substituted macrocycles, is that position of the
benzylic substituent is also shifted one place, from the corner
C1
C2
C3
C4
C5
C6
C7
C8
eq. corner
eq. side
+10.0
+10.8
+0.12
+4.86
+0.36
+7.69
+0.90
+4.81
0.00
3+
of the ring to the side (Figure 8). When the Ln ion drops down
into the coordination cage after deprotonation of the macrocycle,
this positional change of the benzylic substituent is fixed in place,
and a regioisomer with a substituent on the side is produced.
This proposed ligand conformation accounts for the produc-
tion of two regioisomeric chelates during the metalation reac-
tions of NB-DOTMA and 1. It also accounts for the observed pH
and concentration dependence of the product ratios since the
reactions described in Figure 7 and 8 are likely to have different
reaction rates under different conditions. Furthermore, the
mechanism proposed is not restricted to a “two up two down”
ligand conformation; it would also encompass “three up one
down” and “one up three down” conformations. However, we are
able to exclude the involvement of “four up” or “four down”
conformations under these reaction conditions since they would
produce a single regioisomeric product. The involvement of
these latter conformations in the formation of chelates from
unsubstituted macrocycles, such as those of DOTA and DOT-
MA, can not be ruled out since differentiation of the two products
is not possible. It is also possible that they are involved in the
formation of NB-DOTA chelates, although this seems unlikely; it
is more probable that simply the rate of the reaction shown in
Figure 8 is just so slow that it does not contribute to the overall
product distribution.
eq. corner
eq. side
0.00
+14.9
+7.70
+13.1
+0.80
+12.6
eq. corner
eq. side
eq. corner
eq. side
+6.36
a
In the case of H DOTMA the ligand structure was constrained and
6
2
only the energy of the benzylic substituent was minimized; for H DOT-
MA no part of the ligand structure was constrained during the
calculations.
TSAP during chelate formation. Our conventional picture of the
34
ligand during the mechanism of chelate formation is in need of
some refinement.
From the crystallographic data of three stereoisomeric ligands
of TCE-DOTA it is apparent that a number of different ligand
conformations are possible for R-substituted DOTA-type ligand
1
3
systems. Most interestingly, the SSRR- isomer places two
pendant arms “above” the plane of the macrocycle and two
“
below”. To achieve this conformation the helicity of two of the
macrocyclic ethylene bridges are δ while two are λ. The
implications for systems such as NB-DOTMA and 1, should
they adopt comparable conformations, would be significant.
3+
3+
From this starting point the Ln ion may approach the ligand
from either face of the macrocycle and would still encounter
carboxylate ligands with which it could begin the chelation
process. The next step must then be “flipping over” the two
Thermodynamic Stability. Since Ln ions are not well
tolerated in vivo, extremely strong chelates are required for
3+
in vivo applications to ensure that the Ln ion is not released
into the body. Determining the stability constant is an excellent
pendant arms positioned on the opposite face of the macrocyclic
means of assessing the strength of a chelate. The stability
+
+
ring to complete the H LnL chelate. This is readily understood
constants of chelates that form rapidly, such as H GdL vide
2
2
in the situation that gives rise to a chelate with the benzylic
substituent on the corner of the ring (Figure 7). The two pendant
arms to be “flipped” are associated with ethylene bridges in the λ
conformation. Since the macrocycle must adopt a δδδδ con-
formation in the final chelate to accommodate an S- configura-
tion at carbon these two ethylene bridges must also flip
conformations. When this occurs the two pendant arms will also
infra, can be obtained from direct titration of the ligand in the
presence of the metal ion. The complete chelation of Ln ions
3
+
by DOTA-type ligands occurs only very slowly. For these
21
systems a different, out-of-cell, approach is required. The pH
of a series of samples containing ligand and metal are adjusted to
different values in the range of 1.82ꢀ2.90. The samples are then
sealed and incubated at 40 °C for 2 months followed by a further
3 months at room temperature. The pH of each solution is then
recorded. This “out-of-cell” approach allows points on a titration
curve to be determined accurately, and subsequent comparison
with the titration data obtained for the ligand in isolation affords
the stability constant. Stability constants were determined, using
3+
“
flip” onto the other side of the macrocyclic ring thereby
+
completing the H LnL chelate. Subsequent deprotonation of
2
3+
the macrocycle will allow the Ln ion to drop down into the
coordination cage completing the chelation reaction affording a
chelate with the substituent located on the corner.
3
+
3+
3+
However, the Ln ion need not necessarily approach from
that face of the macrocycle. It could equally arrive from the
opposite direction in which case it will begin the chelation
process with those pendant arms associated with the λ ethylene
bridges. The δ ethylene bridges cannot flip their conformation to
bring the remaining pendant arms into position because of the
locking effect of the benzylic substituent. Thus an alternative
route is necessary if this collision is to leads to reaction. The λ
ethylene bridges must flip their conformations since the con-
formations of all ethylene bridges in the final chelate must be the
a 1.0 M KCl ionic background, for the Ce , Gd , and Yb
chelates of S-RRRR-NB-DOTMA (SAP) and S-SSS-1 (TSAP);
the data are collected in Table 7.
3+
The trend most generally observed for the Ln chelates of a
given ligand is that the thermodynamic stability increases as the
3+
ionic radius of the Ln ion becomes smaller. This is easily
understood in terms of an increased electrostatic interaction as
3+
the Ln ions become more charge dense across the series.
However, in the cases of the two ligands under investigation here
an additional factor comes into play. In LnDOTA chelates, the
7
975
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
Figure 7. Conformational changes that are hypothesized to occur during the formation of a chelate that positions the benzylic substituent on the corner of the
macrocycle, shown for the S-SSSS- isomer of NB-DOTMA. The ligand structure is derived from the crystallographic data for RRSS-TCE-DOTA and the
benzylic substituent located in the position found to afford the lowest energy conformation, vide infra. To flip the pendant arms on the left-hand sight of the
macrocycle below the plane of the macrocycle as shown (blue arrows) the conformation of two ethylene bridges must flip λλ to δδ (blue arrows).
Figure 8. Conformational changes that are hypothesized to occur during the formation of a chelate that positions the benzylic substituent on the side of
the macrocycle, shown for the S-SSSS- isomer of NB-DOTMA. The ligand structure is derived from the crystallographic data for RRSS-TCE-DOTA and
the benzylic substituent located in the position found to afford the lowest energy conformation, vide infra. To flip the two pendant arms in the right-hand
side of the ligand above the plane of the macrocycle (as shown) it is necessary for each atom in the macrocycle to undergo a positional shift (green arrow)
which enable the two ethylene bridges in a λ conformation to flip conformations while flipping the pendant arms on the right into a position above the
ring (blue arrows). This positional shift also moves the benzylic substituent from the corner (in the ligand) to the side of the macrocycle (in the chelate).
3
+
SAP and TSAP structures may interconvert freely in solution,
and the distribution of these two isomers is determined by
the relative energy of each. It is well-known that in chelates of
the larger, earlier lanthanides the TSAP isomer, which has a more
open coordination cage, predominates. As one passes along the
series the SAP isomer becomes increasingly more populated as
the ionic radius of the Ln ions decreases, reaching a maximum
3+
when the ionic radius is ∼104 pm. This occurs between Ho and
3+
Er , henceforth, as the ionic radius continues to decrease the
1
4
TSAP isomer becomes increasingly more populated. This
reversal in the trend is apparently a result of the coordinated
water molecule that caps the TSAP structure being pushed off the
14
7
976
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
Table 7. Stability Constants (log K ) of the Ce , Gd , Yb ,
ARTICLE
3
+
3+
3+
ML
3+
and Lu Chelates of S-RRRR-NB-DOTMA (SAP), S-SSS-1
(TSAP), DOTA, and NB-DOTA (I = 1.0 M KCl, 25 °C)
3
+
a
b
Ln
S-RRRR-NB-DOTMA
S-SSS-1
DOTA
NB-DOTA
3
+
Ce
19.79 (0.06)
23.86 (0.07)
24.12 (0.09)
24.04 (0.07)
21.53 (0.11)
21.91 (0.08)
23.4
24.7
25.0
3
+
Gd
24.2
3
+
Yb
3
+
Lu
pGd
24.5
19.51
b
16.02
19.01
19.88
a
From ref 25. From ref 21, data recorded with I = 1.0 M NMe Cl.
4
chelate entirely by the steric demands of a ligand in a TSAP
1
4,35
conformation.
For chelates of both S-RRRR-NB-DOTMA and S-SSS-1 iso-
meric interconversion is not possible; the coordination geometry
is fixed. It is perhaps not surprising then that the stability
constants should follow the trend observed for the SAP/TSAP
isomer distribution in LnDOTA chelates. S-SSS-1, which is
obliged to adopt a TSAP structure, forms its most stable chelate
1
s
Figure 9. H NMR spectra (600 MHz, D O), focusing on the ax
2
resonances, of (a) a sample of YbS-SSS-1 with the nitrobenzyl group
located on the corner of the macrocycle; (b) the same sample after
heating for 336 h at 90 °C and pD1; and (c) a sample of YbS-SSS-1 with
the nitrobenzyl substituent located on the side of the macrocycle that
was isolated from the synthesis of the sample used in (a) and (b).
3
+
with Ce , the largest ion studied. Conversely, the stability
constant of CeS-RRRR-NB-DOTMA, constrained to adopt the
less favorable SAP isomer, is the smallest of any of the chelates
studied and 4.25 log units lower than that of the TSAP isomer. By
the time one reaches GdDOTA the SAP isomer is already the
predominant isomer in solution, constituting g85% of the
sample. Not surprisingly then, the observed stability of GdS-
RRRR-NB-DOTMA was determined to be much higher, ∼4 log
lowest energy configuration at each chiral center and leads to the
eventual production of a single diastereoisomer: the RRRR-/
SSSS- isomer. In the case of nitrobenzyl substituted chelates the
RRRR- and SSSS- isomers are not equivalent, affording SAP and
TSAP isomers, respectively when the configuration at the
macrocycle is S-. This raised the prospect of examining the
relative stability of the two isomers by epimerization. As a
preliminary experiment we examined a sample of YbS-SSS-1 a
chelate constrained to adopt a TSAP geometry despite a
preference of Yb for the SAP geometry. If the R- chiral
centers of this chelate could be induced to epimerize then would
it slowly convert, through other diasteroisomers, from a TSAP
isomer to the more thermodynamically stable SAP isomer?
Accordingly, a 20 mM sample of optically pure chelate was
3+
units higher than that of the corresponding Ce chelate and ∼2
log units higher than that of GdS-SSS-1. The isomer distribution
in YbDOTA is comparable to that of GdDOTA and this too is
reflected in the stability constants of both YbS-RRRR-NB-DOT-
MA and YbS-SSS-1 which are just slightly higher than that
3+
14
3+
observed for the corresponding Gd chelates. The slightly
3+
higher stability constants determined for the Yb chelates,
3
+
relative to the Gd chelates, is then presumably the result of
the aforementioned increased electrostatic interaction between
ligand and metal ion. This observation should not be misinter-
preted as reflecting selectivity for one metal ion over another by
prepared in D O at pD 1 (DCl) and sealed in an NMR tube. The
2
choice of deuterated solvent was critical as it allowed for
verification that epimirization of the R- centers was occurring
by monitoring for deuterium incorporation. The sample was
3
6
each ligand.
It is instructive to compare these stability constants with those
1
then incubated at 90 °C and monitored periodically by H NMR.
25
obtained for LnDOTA chelates. In each case the KLnL for
DOTA closely matches that of whichever chelate was con-
strained to adopt the predominant solution state isomer of
A second chelate was observed to slowly grow in the NMR
spectrum of the sample over a period of several weeks. However,
this new chelate did not exhibit a SAP coordination geometry
(Figure 9), nor did the R-methyne protons (shifted most upfield
and not shown) diminish in signal intensity. From this latter
observation we can conclude that in this chelate no epimerization
of the R- centers occurs under these conditions since there is no
H/D exchange. Consequently, the only means by which a second
chelate structure can be produced is if the metal ion is liberated
from the initial chelate by the acid in the sample, and metal and
ligand then recombine. Under such circumstances two possible
reaction routes are open. First, they can recombine in a reverse
reaction to form the initial chelate. Second, they could undergo a
recombination reaction that shifts the position of the nitrobenzyl
substituent as indicated in Figure 8, thereby producing the
3+
LnDOTA for a given Ln ion. So CeDOTA, in which the TSAP
isomer predominates, has a stability constant that is comparable
to that of CeS-SSS-1 which is constrained to adopt a TSAP
3+
3+
geometry. In the same way the Gd and Yb chelates of both
21
DOTA and NB-DOTA, all predominately SAP, have very
similar stability constants to the S-RRRR-NB-DOTMA chelates
which are constrained into a SAP geometry. It should be noted
that the stability constant data reported herein do not separate
contributions from the two regioisomers (side and corner) of
these chelates discussed above. Each regioisomer is expected to
contribute differently to the overall measurement; however, the
extent of each contribution remains unknown and difficult
to probe.
1
regioisomer of the initial chelate. Figure 9 shows the H NMR
Epimerization Is Not a Route to Isomer Selection. We have
previously shown that chiral centers in the R-position of acetate
pendant arms can be epimerized when the chelate is subjected to
acid, below ∼pH 2. This epimerization will, over time, favor the
spectra of the initial chelate YbS-SSS-1 in which the nitrobenzyl
group is located on the corner of the macrocycle, the same
sample after 336 h of heating at pD 1 and 90 °C and the sample of
a YbS-SSS-1 in which the nitrobenzyl group is located on the side
1
3
7
977
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
ARTICLE
how the ligand rearranges both the pendant arms and the
+
ethylene bridge conformations to form the H LnL intermedi-
2
ate. The involvement of this intermediate has been confirmed
allowing rate constants for the final rate determining step in the
chelate formation. As for other related chelates the value of k
H O
2
for these nitrobenzyl ligands is small indicating that a base
catalyzed pathway predominates. The rate constants for this
process kOH are found to be much smaller in cases where the
ꢀ
ligand system is substituted on the pendant arms, much more so
than substitution of the macrocycle. This indicates that con-
formational motion of the macrocycle plays little or no part in
3
+
allowing the Ln ion to drop down into the coordination cage.
In contrast substitution of the pendant arms, which equates to
restricted conformational motion of these arms, does appear to
be an important factor in the deprotonation and rearrangement
Figure 10. Shown are the decreasing proportion YbS-SSS-1 with the
nitrobenzyl located on the corner of the macrocycle (red circles) and
the corresponding increase in the proportion of YbS-SSS-1 with the
nitrobenzyl group located on the side of the macrocycle (blue
+
of H LnL to the fully formed chelate.
2
The chelates produced by these conformationally restricted
ligands are stable, of comparable stability to the corresponding
DOTA chelates. An interesting relationship is observed between
1
diamonds) as determined by H NMR as a sample of YbS-SSS-1
3+
(with the nitrobenzyl group located on the corner of the macrocycle) is
the stability constants, the size of the Ln ion, and the nature of
the ligand. The thermodynamic stability of a chelate is highest for
DOTA-type ligands when the ligand preferentially adopts the
coordination geometry most preferred by that metal ion, that is,
heated at pD1 and 90 °C.
of the macrocycle. This last sample was isolated as a side
product from the reaction that produced the sample with the
substituent on the corner. From these NMR spectra it is clear
that the position of the nitrobenzyl substituent is being
scrambled during this process to afford a mixture of chelates
in which the nitrobenzyl substituent is either located on the
corner or the side of the macrocyclic ring. The development of
this latter regioisomer over time is shown in Figure 10, which
indicates that an equilibrium point will eventually be reached.
Although the position of this equilibrium point is undoubtedly
dependent upon the pH and presumably also the reaction
temperature, in this case it lies close to 50/50. Further studies
would be required to probe whether this end point is the result
of thermodynamics or the kinetics of the various reaction
processes involved.
3+
early Ln ions are more stable with S-SSS-1 (a TSAP ligand)
3+
whereas later Ln ions preferred the S-RRRR-DOTMA (SAP
ligand). From the data presented herein it seems apparent that
these conformationally restricted chelates are stable enough for
in vivo applications. However, the production of multiple
regioisomers during metalation is a problem that may well prove
intractable and may limit the desirability of this particular system
to molecular imaging applications.
’ ASSOCIATED CONTENT
S
Supporting Information. Crystallographic data in CIF
b
format. Further details are given in Figures S1ꢀS6. This material
is available free of charge via the Internet at http://pubs.acs.org.
’
CONCLUSIONS
’ AUTHOR INFORMATION
Our previously reported conclusions regarding the chelates
Corresponding Author
*
mark.woods@pdx.edu or woodsmar@ohsu.edu.
obtained when preparing NB-DOTMA chelates were mistaken.
Although the amine in the 1-position of the macrocycle is
undoubtedly a poorer nucleophile than those in the other
positions of the ring, it is sufficiently powerful to undertake
Phone: + 1 503 725 8238 or + 1 503 418 5530. E-mail:
an S 2 reaction. Thus, the alkylation reaction produces a single
N
’
ACKNOWLEDGMENT
ligand stereoisomer; however, when a chelate is formed from
this single stereoisomer two regioisomeric chelates may result.
The distribution of these regioisomeric chelates produced is
dependent upon the reaction conditions employed during
metalation. This observation of the formation of regioisomeric
chelates may also explain the production of multiple isomeric
The authors thank the National Institutes of Health (EB-
011687), Oregon Nanoscience and Microtechnologies Insti-
tute (N00014-11-1-0193); a J ꢀa nos Bolyai Research Scholarship
of the Hungarian Academy of Sciences, the Hungarian Scien-
ꢀ
tific Research Found (OTKA K84291) and the TAMOP 4.2.1./
2
+
37
chelates in the related Cu chelates of NB-NOTA. We
hypothesize that the production of more than one regioisomer
during the chelation process stems from the conformation of
B-09/1/KONV-2010-0007 project implemented through the
New Hungary Development Plan, cofinanced by the European
Social Fund and the European Regional Development Fund
(G.T.); the M.J. Murdock Charitable Trust, Portland State
University and the Oregon Opportunity for biomedicine for
financial support of this work and the National Science Foun-
dation (0741993) for funding the purchase of the LTQ-Orbi-
trap Discovery. This research was performed within the
framework of the EU COST Action D38 “Metal-Based Systems
for Molecular Imaging Applications”.
the ligand in solution before the formation of the intermediate
+
H LnL complex. This conformation must involve placing
2
pendant arms on either side of (above and below) the macro-
cyclic ring. It also would seem likely that a mixture of δ and λ
conformations is present for the ethylene bridges within each
macrocyclic ring. The regioisomer obtained is thus dependent
upon the face from which the metal approaches the ligand and
7
978
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979

Inorganic Chemistry
REFERENCES
ARTICLE
’
(31) Moreau, J.; Guillon, E.; Pierrard, J.-C.; Rimbault, J.; Port, M.;
Aplincourt, M. Chem.—Eur. J. 2004, 10 (20), 5218–5232.
(
3
(
1) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem.
Rev. 1999, 99 (9), 2293–2352.
2) Xu, J.; Franklin, S. J.; Whisenhunt, D. W., Jr.; Raymond, K. N.
J. Am. Chem. Soc. 1995, 117 (27), 7245–7246.
3) Burai, L.; Toth, E.; Merbach, A. E. Chem. Commun. 2003, 21,
680–2681.
4) Laus, S.; Ruloff, R.; Toth, E.; Merbach, A. E. Chem.—Eur. J.
003, 9 (15), 3555–3566.
5) Kotek, J.; Lebduskova, P.; Hermann, P.; Van der Elst, L.; Muller,
32) Wu, S. L.; Horrocks, W. D., Jr. Inorg. Chem. 1995, 34 (14),
724–3732.
33) Wang, X.; Jin, T.; Comblin, V.; Lopez-Mut, A.; Merciny, E.;
Desreux, J. F. Inorg. Chem. 1992, 31 (6), 1095–1099.
34) Sherry, A. D.; Caravan, P.; Lenkinski Robert, E. Magn. Reson.
Imaging 2009, 30 (6), 1240–1248.
35) Benetollo, F.; Bombieri, G.; Calabi, L.; Aime, S.; Botta, M. Inorg.
Chem. 2003, 42 (1), 148–157.
36) Di Bernardo, P.; Zanonato, P. L.; Bismondo, A.; Melchior, A.;
Tolazzi, M. Dalton Trans. 2009, 21, 4236–4244.
37) Schlesinger, J.; Rajander, J.; Ihalainen, J. A.; Ramesh, D.;
Eklund, P.; Fagerholm, V.; Nuutila, P.; Solin, O. Inorg. Chem. 2011, 50
(
(
(
(
2
(
(
2
(
(
R. N.; Geraldes, C. F. G. C.; Maschmeyer, T.; Lukes, I.; Peters, J. A.
Chem.—Eur. J. 2003, 9 (23), 5899–5915.
(
(
6) Woods, M.; Kovacs, Z.; Zhang, S.; Sherry, A. D. Angew. Chem.,
Int. Ed. 2003, 42 (47), 5889–5892.
7) Woods, M.; Botta, M.; Avedano, S.; Wang, J.; Sherry, A. D.
Dalton Trans. 2005, 24, 3829–3837.
8) Rudovsky, J.; Cigler, P.; Kotek, J.; Hermann, P.; Vojtisek, P.;
Lukes, I.; Peters, J. A.; Vander Elst, L.; Muller, R. N. Chem.—Eur. J. 2005,
(10), 4260–4271.
(
(
1
1 (8), 2373–2384.
9) Aime, S.; Botta, M.; Ermondi, G. Inorg. Chem. 1992, 31 (21),
291–4299.
(
4
(10) Hoeft, S.; Roth, K. Chem. Ber. 1993, 126 (4), 869–873.
(11) Aime, S.; Barge, A.; Bruce, J. I.; Botta, M.; Howard, J. A. K.;
Moloney, J. M.; Parker, D.; de Sousa, A. S.; Woods, M. J. Am. Chem. Soc.
1
999, 121 (24), 5762–5771.
12) Dunand, F. A.; Aime, S.; Merbach, A. E. J. Am. Chem. Soc. 2000,
22 (7), 1506–1512.
13) Woods, M.; Aime, S.; Botta, M.; Howard, J. A. K.; Moloney,
J. M.; Navet, M.; Parker, D.; Port, M.; Rousseaux, O. J. Am. Chem. Soc.
000, 122 (40), 9781–9792.
14) Aime, S.; Botta, M.; Fasano, M.; Marques, M. P. M.; Geraldes,
C. F. G. C.; Pubanz, D.; Merbach, A. E. Inorg. Chem. 1997, 36 (10),
059–2068.
15) Aime, S.; Botta, M.; Garda, Z.; Kucera, B. E.; Tircso, G.; Young,
V. G.; Woods, M. Inorg. Chem. 2010, DOI: 10.1021/ic2012827.
16) SMART, V5.054; Bruker Analytical X-ray Systems: Madison,
WI, 2001.
(
1
(
2
(
2
(
(
(
(
17) Blessing, R. H. Acta Crystallogr. 1995, A51 (1), 33–8.
18) SAINT+, V6.45; Bruker Analytical X-Ray Systems: Madison,
WI, 2003.
(
19) SHELXTL, V6.14; Bruker Analytical X-Ray Systems: Madison,
WI, 2000.
(
20) Irving, H. M.; Miles, M. G.; Pettit, L. D. Anal. Chim. Acta 1967,
38 (4), 475.
(
21) Woods, M.; Kovacs, Z.; Kiraly, R.; Br €u cher, E.; Zhang, S.;
Sherry, A. D. Inorg. Chem. 2004, 43 (9), 2845–2851.
22) Z ꢀe k ꢀa ny, L.; Nagyp ꢀa l, I. Computational Methods for Determi-
(
nation of Formation Constants. In Computational Methods for Determi-
nation of Formation Constants; Legget, D. J., Ed.; Kluwer: New York,
1
985; p 291.
23) Meyer, M.; Dahaoui-Gindrey, V.; Lecomte, C.; Guilard, R.
Coord. Chem. Rev. 1998, 178ꢀ180 (Pt. 2), 1313–1405.
24) Di Bari, L.; Pescitelli, G.; Sherry, A. D.; Woods, M. Inorg. Chem.
005, 44 (23), 8391–8398.
25) Cacheris, W. P.; Nickle, S. K.; Sherry, A. D. Inorg. Chem. 1987,
6 (6), 958–960.
26) Hancock, R. D.; Motekaitis, R. J.; Mashishi, J.; Cukrowski, I.;
Reibenspies, J. H.; Martell, A. E. J. Chem. Soc., Perkin Trans. 2 1996, No.
, 1925–1929.
(
(
2
(
2
(
9
(27) Delgado, R.; da Silva, J. J. Talanta 1982, 29 (10), 815–822.
(28) Kalman, F. K.; Woods, M.; Caravan, P.; Jurek, P.; Spiller, M.;
Tircso, G.; Kiraly, R.; Brucher, E.; Sherry, A. D. Inorg. Chem. 2007, 46
(13), 5260–5270.
(
29) Smith, R. M.; Martell, A. E. Sci. Total Environ. 1987, 64 (1ꢀ2),
25–147.
30) T ꢀo th, E.; Br €u cher, E.; L ꢀa z ꢀa r, I.; T ꢀo th, I. Inorg. Chem. 1994, 33
18), 4070–4076.
1
ꢀ
(
(
7
979
dx.doi.org/10.1021/ic2012843 |Inorg. Chem. 2011, 50, 7966–7979