Synthesis and relaxivity studies of a gadolinium(III) complex of ATP-conjugated DO3A as a contrast enhancing agent for MRI

Article abstract of DOI:10.1002/ejic.200401018

A gadolinium(III) complex of adenosine 5′-triphosphate (ATP)-appended DO3A has been synthesized in order to attain higher relaxivity and to reduce the in vivo toxicity. DO3A (2) was synthesized as the exclusive product by a single step direct trialkylation of cyclen with chloroacetic acid in water (pH 10, -4°C). ATP was then covalently appended to the DO3A framework through a propyl linker. The 3-bromopropane spacer appended DO3A (3) was synthesized by the reaction of DO3A with 1,3-dibromopropane in water/DMF in the presence of triethylamine as proton scavenger. The ATP-appended DO3A (DOSA-Pr-ATP) was synthesized by the reaction of 3 with ATP in water at room temperature. [Gd(DOSA-Pr-ATP)(H₂O)₂] (4) was synthesized by the reaction of DOSA-Pr-ATP with gadolinium(III) perchlorate hydrate in water. The X- and Q-band EPR spectra of 4 contain a broad band with no hyperfine splitting at both room temperature and liquid nitrogen temperature. The g-values are 2.167 and 2.033 at X- and Q-band, respectively. The magnetic moment of 4 is 7.45 BM which is close to the value for free Gd^III ion. The longitudinal relaxivity, r_1p, of [Gd(DO3A-Pr-ATP)(H₂O)₂] is 6.51 mM^-1s^-1 (24 MHz and 35±0.1°C), which is higher than that of [Gd(DOTA)(H₂O)]^- (r_1p = 3.50 mM^-1s^-1, 20 MHz, 39°C, pH 7.3) and [Gd(DO3A)-(H₂O)₂] (r_1p = 4.8 mM ^-1s^-1, 20 MHz, 40°C). The higher relaxivity of 4 than for other systems with q = 2 is due to the increase in the molecular dimension of the complex by the conjugation of ATP. The relaxivity of 4 at pH 8.4 (TRIS buffer) decreases to 5.64 mM^-1s^-1, probably due to a change in the hydration number by the replacement of the coordinated water molecules by TRIS. The r_1p relaxivity of 4 in the presence of β-cyclodextrin is 8.97 mM^-1s^-1 due to the increase in the molecular weight and dimension of the inclusion complex formed by the noncovalent host-guest interaction of the ATP pendant arm with the hydrophobic cavity of β-cyclodextrin. The transverse relaxivity, r_2p, of 4 is 7.48 mM^-1s^-1 (24 MHz and 35±0.1°C). The r_2p/r_1p ratio of 1.16 indicates that 4 is a T ₁-weighted contrast agent. The ATP moiety remains as an extended pendant arm and does not bind with the metal ion, nor does it block the coordination sites for the inner-sphere water molecules. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejic.200401018

FULL PAPER
Synthesis and Relaxivity Studies of a Gadolinium(III) Complex of ATP-
Conjugated DO3A as a Contrast Enhancing Agent for MRI
[a]
[a]
S. James Ratnakar and Vedhamonickom Alexander*
Keywords: Gadolinium / Imaging agents / Macrocyclic ligands / N ligands
A
(
gadolinium(III
ATP)-appended DO3A has been synthesized in order to at-
tain higher relaxivity and to reduce the in vivo toxicity. DO3A
2) was synthesized as the exclusive product by a single step
direct trialkylation of cyclen with chloroacetic acid in water
pH 10, –4 °C). ATP was then covalently appended to the
)
complex of adenosine 5Ј-triphosphate
which is higher than that of [Gd(DOTA)(H
2
O)]– (r1p
s , 20 MHz, 39 °C, pH 7.3) and [Gd(DO3A)-
=
–
1 –1
3.56 m
M
–
1 –1
(H O)
2
2
] (r1p = 4.8 m
M
s , 20 MHz, 40 °C). The higher re-
(
laxivity of 4 than for other systems with q = 2 is due to the
increase in the molecular dimension of the complex by the
conjugation of ATP. The relaxivity of 4 at pH 8.4 (TRIS buffer)
(
–
1 –1
DO3A framework through a propyl linker. The 3-bromopro- decreases to 5.64 m
M
s , probably due to a change in the
pane spacer appended DO3A (3) was synthesized by the re-
action of DO3A with 1,3-dibromopropane in water/DMF in
the presence of triethylamine as proton scavenger. The ATP-
hydration number by the replacement of the coordinated
water molecules by TRIS. The r1p relaxivity of 4 in the pres-
–
1 –1
ence of β-cyclodextrin is 8.97 m
M
s
due to the increase in
appended DO3A (DO3A-Pr-ATP) was synthesized by the re- the molecular weight and dimension of the inclusion com-
action of
3
with ATP in water at room temperature.
O) ] (4) was synthesized by the reac-
plex formed by the noncovalent host–guest interaction of the
[Gd(DO3A-Pr-ATP)(H
2
2
ATP pendant arm with the hydrophobic cavity of β-cyclodex-
–
1 –1
tion of DO3A-Pr-ATP with gadolinium(III) perchlorate hy-
drate in water. The X- and Q-band EPR spectra of 4 contain
a broad band with no hyperfine splitting at both room tem-
perature and liquid nitrogen temperature. The g-values are
trin. The transverse relaxivity, r2p, of 4 is 7.48 m
(24 MHz and 35±0.1 °C). The r2p/r1p ratio of 1.16 indicates
that 4 is a T -weighted contrast agent. The ATP moiety re-
M
s
1
mains as an extended pendant arm and does not bind with
the metal ion, nor does it block the coordination sites for the
inner-sphere water molecules.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
2.167 and 2.033 at X- and Q-band, respectively. The mag-
netic moment of 4 is 7.45 BM which is close to the value for
III
free Gd ion. The longitudinal relaxivity, r1p, of [Gd(DO3A-
–
1 –1
Pr-ATP)(H
2
O)
2
] is 6.51 m
M
s
(24 MHz and 35±0.1 °C),
Introduction
verse relaxation rates of the water protons which contribute
to the intensity of the MRI signal and thus to the contrast
of the image. The first example of a paramagnetic substance
used for tissue discrimination in MRI was provided by Lau-
MRI is a state-of-the-art, noninvasive imaging modality
in clinical medicine, as exemplified by the award of the 2003
Nobel Prize for medicine to Professor Paul C. Lauterbur
[3]
terbur et al. The in vitro and in vivo chemical results re-
(Biomedical Magnetic Resonance Laboratory, University of
[4]
ported by Tweedle et al. support the usefulness of strongly
Illinois) and Professor Sir Peter Mansfield (University of
Nottingham) for their seminal discoveries concerning the
use of magnetic resonance to visualize different structures
leading to the development of modern MRI.[1] An MRI
image is a three dimensional signal intensity map of the
spatially encoded proton signal of the in vivo water mole-
cules in a given volume element (voxel). The movement of
protons from one position to another during the relaxation
experiment alters the signal intensity and therefore the mov-
ing blood has no detectable signal. It is common to increase
the contrast of the image by administering MRI contrast
III
III
chelated Gd complexes as CAs, and since then Gd com-
plexes of polyaminocarboxylate ligands have been widely
[2a,2e,2f]
studied.
III
Proton relaxivity by Gd -based CAs is governed by the
magnetic coupling interaction of the unpaired electrons of
the metal ion and the water proton nuclei by a through-
[5]
space dipole–dipole mechanism and is interpreted using
[6]
the Solomon–Bloembergen–Morgan (SBM) theory. Re-
[5d,7]
laxivity is determined by several parameters,
and higher
relaxivity may be obtained by increasing the correlation
time, τ , which modulates the dipole–dipole relaxation
c
[
2]
agents (CAs), which enhance the longitudinal and trans-
mechanism. It depends on the exchange lifetime (kex
/τ ) of the coordinated water molecules, the electronic re-
=
1
m
[
a] Department of Chemistry, Loyola College,
laxation times (T1e,2e), and the rotational correlation time
Chennai 600034, India
(
τR) of the complex. One of the practical approaches to
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
gain higher relaxivity by gadolinium-based contrast agents
3918
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejic.200401018 Eur. J. Inorg. Chem. 2005, 3918–3927

Gadolinium() Complex as a Contrast Enhancing Agent for MRI
FULL PAPER
is increasing τ by increasing the size and molecular weight image contrasts even at a lower concentration than nor-
R
III
of Gd chelates by covalent or noncovalent interaction mally required. DO3A is a well-suited ligand framework
III
with macromolecules. Examples of Gd chelates bound to for functionalization due to the unique secondary amine
macromolecules include the HSA adduct of the Gd com- nitrogen of the macrocyclic ring. Furthermore, biomacro-
plex of benzyloxy-α-propionic acid substituted DOTA (r
molecule-appended DO3A is expected to exhibit the same
1
p
–
1 –1 [8]
=
56 m s ), the Gd complex of a p-bromobenzyloxy- stability as that of the parent DO3A. Herein we report the
–
1 –1
substituted pyridine macrocycle (r = 8.25 m s ), its synthesis and relaxivity studies of gadolinium() complex
HSA adduct (r1p = 48 m s ), and its noncovalent adduct of adenosine 5Ј-triphosphate appended DO3A.
with poly-β-cyclodextrin (r1p = 36 m s ), the squaric
ester substituted DO3A linked to polyornithine (r
1
p
–
1 –1
–
1 –1 [9]
=
p
1
–
1
–1 [10]
[11]
–1 –1
29 m s ),
Gd-DTPA-polylysine
(r1p = 13 m s
Results and Discussion
–
1 –1
and 32 m s at pH 8), the Gd dendrimer of the DTPA-
functionalized second-generation starburst dendrimer (r
=
1
p
Synthesis of the Ligand
–
1
–1
[12]
234 m s at 25 MHz),
the Gd complex of p-ni-
–
1 –1
trophenyl-substituted DOTA (r = 5.4 m s , 20 MHz,
1
p
1
(
,4,7-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
DO3A) (2)
[
13]
2
5
5 °C, pH 7.3),
0.8 m s , 20 MHz),
the HSA adduct of MS-325 (r1p =
–
1
–1
[14]
the glucitol-conjugated DO3A
–
1
–1 [15]
DO3A was synthesized in a single-step direct N-alky-
(r1p = 5.19 m s ), Gd chelates of p-bromobenzyl- and
lation of cyclen with chloroacetic acid in a 1:3 molar ratio
in 69.58% yield. The reaction was carried out at –4 °C and
gradually heated to 50 °C over 4 h. The pH of the medium
was maintained at 10 throughout the reaction. DO3A was
p-phosphonatomethylbenzanilido-substituted DO3A (r
=
p
1
–
1
–1
–1 –1
7
.9 m s and 10.7 m s , respectively), and their HSA
–
1
–1
–1 –1
adducts (r = 24.0 m s and 21.0 m s , respec-
tively),
attached to a polysaccharide backbone (r = 14.4 m s
1
p
[
16]
and the multiple Gd-DO3A chelates covalently
[
30]
–
1
–1
first synthesized by Tweedle et al. by the reaction of cy-
clen with chloroacetic acid in water at pH 10. The reaction
afforded both DO3A and DOTA and the separation was
carried out by ion-exchange chromatography. Tweedle et
1
p
for each Gd, 20 MHz, 39 °C).[
17]
The gadolinium() chelates approved by the FDA for
[
18]
human imaging are (NMG) [Gd(DTPA)(H O)]
(Mag-
(Dotarem,
2
2
[
21a]
[
19]
al.
have also reported the synthesis of DO3A by the al-
nevist, Schering), (NMG)[Gd(DOTA)(H O)]
2
[20]
kylation of monoprotected cyclen (p-toulenesulfonyl or for-
myl group) with chloroacetic acid followed by deprotection.
Guerbet), [Gd(DTPA-BMA)(H O)]
(Omniscan, Ny-
(ProHance, Bracco),
(Gadovist, Schering), and
(MultiHance, Bracco). New classes
2
[21]
comed), [Gd(HP-DO3A)(H O)]
2
22]
[
[
[
Gd(DO3A)-Butrol)(H O)]
2
23]
Linker-Appended DO3A (3)
[
Gd(BOPTA)(H O)]
2
[24]
[25]
DO3A is a heptadentate ligand with three acetate arms
and a unique secondary amine nitrogen on the ring. The
bromopropyl linker appended DO3A (3) was synthesized
by the N-alkylation of DO3A with 1,3-dibromopropane in
the presence of triethylamine as proton scavenger in DMF/
water at room temperature. The use of triethylamine as a
base leads to the formation of its hydrobromide salt along
with 3, which is removed by washing with methanol. The
reaction is illustrated in Scheme 1.
of MRI contrast agents
targeting CAs,
that are pH-sensitive,
such as blood pool agents,
and smart contrast agents such as those
[
24]
[10,25i,26]
oxygen-pressure (pO ) respon-
2
[
27]
[28]
sive,
enzyme responsive,
and metal ion concentration
[
29]
dependent are now emerging.
Clinical MRI applications continue to evolve rapidly.
This drives the need for more efficient and pathology-spe-
cific contrast agents that will increase the potential of MRI
as a diagnostic tool. Increasing the molecular weight of a
III
Gd chelate with two inner-sphere water molecules by con-
ATP-Appended DO3A (DO3A-Pr-ATP)
jugating with a biomacromolecule would lead to a biocom-
patible CA with high relaxivity. Such chelates with high tis-
ATP was appended onto the DO3A framework through
sue-specific targeting moieties are able to provide improved the bromopropyl linker by the reaction of 3 with ATP in a
2
Scheme 1. Synthesis of linker appended DO3A. Reagents and conditions: (a) –4 °C, H O, NaOH, pH 10; (b) 1,3-dibromopropane, DMF/
water, triethylamine.
Eur. J. Inorg. Chem. 2005, 3918–3927
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3919

S. J. Ratnakar, V. Alexander
FULL PAPER
Scheme 2. Synthesis of DO3A-Pr-ATP. Conditions: (a) water, room temp., 36 h.
1:1 molar ratio in water at room temperature (Scheme 2). Spectroscopic Studies
The reaction of 3 with the commercially available disodium
salt of ATP yielded DO3A-Pr-ATP along with sodium bro-
mide as the side product. Since both are soluble in water
Infrared Spectrum
The IR spectrum of DO3A-Pr-ATP shows a broad peak
the isolation of the free ligand becomes difficult. Therefore, at 3420.1 cm–1 assignable to the ν (OH) vibration. The
s
–1
the free form of ATP, recovered from its disodium salt by peaks at 2926.5 and 1476.8 cm are assignable to the
passing an aqueous solution through a cation exchange ν (CH) and δ (CH) vibrations, respectively. The sharp peaks
s
s
resin and eluting with water, was reacted with 3. DO3A-Pr- at 1700 and 1635.4 cm–1 are due to the ν (C=O) and δ (NH)
s
s
–1
ATP was isolated as a white solid by adding methanol.
vibrations, respectively. The sharp peak at 1507.1 cm is
Figure 1. ESI mass spectrum of 10-[3-(adenosine 5Ј-triphosphate)oxypropyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
DO3A-Pr-ATP).
(
3920
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2005, 3918–3927

Gadolinium() Complex as a Contrast Enhancing Agent for MRI
FULL PAPER
assigned to the ν (C–O) vibration of the ether linkage be- at δ = 52.99 ppm is assignable to the ethylenic carbon of the
s
tween the DO3A and ATP. The peak at 1087.6 cm–1 is as- macrocyclic framework. The resonance at δ = 57.23 ppm is
signed to the ν (C–N) vibration. The bands at 1228.4 and assignable to the methylene carbon of the acetate arm.
s
–
1
9
00 cm are assigned to the ν (P=O) and ν (P–O) vi-
brations, respectively.
s s
31P NMR Spectrum
[31]
The IR spectrum of the complex
The 31P NMR spectrum of DO3A-Pr-ATP in D
O shows
contains the characteristic absorption bands of the ligand
2
except for the OH vibrations of the carboxylic acid. The three distinct signals due to the three phosphate groups of
–1
band at 1608.8 cm in the complex is typical of the COO ATP in different environments. The signal at δ =
[
32]
functionality.
130.20 ppm is assigned to the central phosphate and the
signal at δ = 107.35 ppm is assignable to the phosphate at-
tached to the propyl spacer. The signal at δ = 119.30 ppm
ESI Mass Spectrum
The ESI mass spectrum of DO3A-Pr-ATP shows a peak is assigned to the phosphate that is attached to the furan
+
at m/z
=
894 for the molecular ion [M
+
1]
group of ATP.
(
C H N O P ). The peak at m/z = 756 is assignable to
2
7
47
9
19 3
+
Mass Spectrum of [Gd(DO3A-Pr-ATP)(H O) ]
2 2
the species [M – C H N ] (C H N O P ) formed by the
5
7
5
22 39
4
19 3
loss of the adenine moiety of ATP. The peak at m/z = 654
The ESI mass spectrum shows a cluster of peaks at m/z
= 1081 assignable to the molecular ion [M – 2]
+
+
is assignable to the species [M
–
C H N O ]
9
13
5
3
(
C H N O P ) formed by the loss of the furan moiety of [Gd(C27
H
41
N
9
O
19
P
3
)(H
2
O)
2
]. The peak at m/z = 793 is as-
1
8
33
4
16 3
+
ATP. The peak at m/z = 348 is assignable to the species signable to [M – C11
H N O ] [Gd(C17H N O P )], the
17 5 5 27 4 16 3
+
[
M – C H N O P ] (C H N O ) formed by the loss of species formed by the loss of the adenine moiety. The peak
13 19 5 13 3 14 28 4 6
+
the phosphate group of ATP and the propyl linker group. at m/z = 734 is assignable to the species [M – C12
The ESI mass spectrum of DO3A-Pr-ATP is depicted in [Gd(C15 )] formed by the loss of the acetate
group. The peak at m/z = 578 is assignable to the species
H N O ]
21 5 7
H N O P
24 4 14 3
Figure 1.
+
[
M – Gd(C H N O )] (C H N O P ) formed by the
12 20 5 7 15 25 4 14 3
III
1H NMR Spectrum
loss of Gd . The peak at m/z = 483 is assignable to [M –
1
+
The H NMR spectrum of DO3A-Pr-ATP in D O shows Gd(C12
H
N
O
11P)] (C15
H
N
O
P
), the species formed
2
20
5
25
4
10
2
resonances for the methylene protons of the cyclen frame- by the loss of one phosphate group. The peak at m/z = 168
+
work as a multiplet at δ = 3.10–3.4 ppm (12 H, is assignable to [M – Gd(C19
H N O P )] (C H N ), the
29 5 21 3 8 16 4
NCH CH N). The triplet at δ = 3.58–3.63 ppm (J = 5.5 Hz, species formed by the loss of acetate groups and the propyl
2
2
2
H) is assigned to the OCH protons of the linker that is linker. The MALDI-TOF mass spectrum shows a peak at
2
bonded to ATP. The resonance at δ = 4.37 ppm (6 H, m/z = 1043.80 (100%) assignable to the molecular ion [M –
+
CH COO) is assigned to the methylene protons of acetate 2H
O] [Gd(C27
H N O P )], which further confirms the
41 9 19 3
2
2
arms. The triplet centered at δ = 4.25 ppm (1 H) is assigned formation of the complex. The MALDI TOF mass spec-
to the proton of the furan ring of the adenine moiety. The trum of [Gd(DO3A-Pr-ATP)(H
O) ] is given in Figure 3.
2
2
doublet at δ = 4.49–4.65 ppm (2 H) is assigned to the OCH2
EPR Spectra and Magnetic Moment of [Gd(DO3A-Pr-
ATP)(H O) ]
protons of the ATP unit. The signals at δ = 2.25 ppm are
assigned to the protons of the propyl linker. The resonances
at δ = 8.37, 8.54 (m, 2 H), and 6.07 ppm (m, 1 H) are as-
2
2
The X- and Q-band EPR spectra consist of a broad band
signed to the aromatic protons of the adenine moiety. The with no hyperfine splitting at room temperature or at liquid
1
H NMR spectrum of DO3A-Pr-ATP is depicted in Fig- nitrogen temperature. The g-value at X-band is 2.167 and
ure 2.
at Q-band is 2.0333. The g-value at 77 K is 2.103 (X-band).
The broad band shows that the dipolar interaction of the
metal ion is small. The magnetic moment of [Gd(DO3A-
13C NMR Spectrum
The 13C NMR spectrum of DO3A-Pr-ATP in D O has Pr-ATP)(H
O) ] is 7.5 BM which is close to the value for
2 2
2
a signal at δ = 174.52 ppm assignable to the carboxylic acid the free gadolinium() ion (7.85 BM).
carbon. The signals at δ = 152.90–145.57 ppm are assign-
able to the carbon atoms of the adenine group of ATP. The
Relaxivity Studies
resonances at δ = 68.43 (OCH ), 77.38 (OCH ), and
2
2
8
7.14 ppm are assignable to the C , C , and C carbons,
2 3 4
Longitudinal Relaxivity (r )
1p
respectively, of the furan ring. The signal at δ = 34.78 ppm
is assignable to the β-CH carbon of the propyl linker. The
The ligand DO3A-Pr-ATP has the DO3A framework
2
resonance at δ = 52.99 ppm is due to the methylene carbon with an extended ATP pendant arm. It coordinates to the
of the propyl linker bonded to the nitrogen of the macrocy- metal ion through four nitrogen donors of the macrocycle
cle. The signal at δ = 68.43 ppm is assigned to the methylene and three carboxylate oxygens. Two inner-sphere water
carbon attached to the phosphate group of ATP. The higher molecules complete the coordination geometry. The com-
chemical shift of this carbon compared to the other two plex [Gd(DO3A-Pr-ATP)(H O) ] (4) is expected to have the
2
2
methylene carbons of the linker is indicative of its attach- same coordination geometry as that of [Gd(DO3A)(H O) ].
2
2
–1 –1
ment to the terminal phosphate group of ATP. The signal The longitudinal relaxivity of 4 is 6.51 m s (normal-
Eur. J. Inorg. Chem. 2005, 3918–3927
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3921

S. J. Ratnakar, V. Alexander
FULL PAPER
Figure 2. 1H NMR spectrum of 10-[3-(adenosine 5Ј-triphosphate)oxypropyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane
DO3A-Pr-ATP).
(
2 2
Figure 3. MALDI TOF mass spectrum of [Gd(DO3A-Pr-ATP)(H O) ] (4).
–
1
–1
[15]
ized to 1 m). The contribution by the inner-sphere mecha- 3.56 m s , 20 MHz, 39 °C, pH 7.3),
a q = 1 system,
–1
–1
–1 –1
nism to the overall relaxivity is approximately 1.7 m s
and [Gd(DO3A)(H O) ] (r = 4.8 m s at 20 MHz,
2 2 1p
per coordinated water molecule.[
2a,2d]
The higher longitudi- 40 °C), a q = 2 system, is attributed not only to the pres-
[33]
–
nal relaxivity of 4 than that of [Gd(DOTA)(H O)] (r
=
ence of two inner-sphere water molecules bound to the me-
2
1p
3922
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2005, 3918–3927

Gadolinium() Complex as a Contrast Enhancing Agent for MRI
FULL PAPER
tal ion, but also to the longer rotational correlation time,
τR, due to the increase in the molecular dimensions of the
complex by the conjugation of ATP. As the molecular tum-
bling rate is reduced by increasing the molecular dimension,
the dipole–dipole interaction between the paramagnetic me-
tal ion and the water proton is efficient and, as a result, a
greater paramagnetic effect is transferred to the bulk sol-
[34]
vent (water). Tweedle et al. have shown that the relaxivity
III
of Gd complexes increases with increasing molecular
weight. The outer-sphere contribution to overall relaxivity
–
1 –1
is approximately 2.3–2.5 m s deduced on the basis of
III
the relaxivity of Gd complexes with no inner-sphere water
molecules (q = 0).
[
35]
Figure 4. A plot of concentration of [Gd(DO3A-Pr-ATP)(H O) ]
2
2
–
1
vs. T
1
in the presence (᭹) and in the absence (᭡) of β-cyclodex-
trin.
Longitudinal Relaxivity in the Presence of β-Cyclodextrin
The longitudinal relaxivity of [Gd(DO3A-Pr-ATP)(H O) ]
2
2
–
1
–1
is due to the competition of the hydroxide ion for coordina-
tion to the Gd ion. The decrease in the relaxivity of
increases to 8.97 m s in the presence of β-cyclodex-
trin. This is attributed to the formation of an inclusion
complex through a nonbonding interaction between the
ATP pendant arm and β-cyclodextrin with a concomitant
III
[
Gd(DO3A-Pr-ATP)(H O) ] at higher pH may be due to
2 2
the replacement of bound water molecules by TRIS.
increase in the molecular dimension and τ of the complex,
R
Transverse Relaxivity (r )
2p
which leads to an enhancement in relaxivity. An aqueous
solution of β-cyclodextrin (50 m) under the same experi-
mental conditions does not show enhancement of water
proton relaxivity. The concentration of β-cyclodextrin was
maintained at 50-times the concentration of the complex to
ensure the formation of the inclusion complex. Aime and
The transverse relaxivity of [Gd(DO3A-Pr-ATP)(H O) ]
2
2
–1 –1
is 7.48 m s (24 MHz and 35± 0.1 °C), which is higher
–
–1 –1
than that of [Gd(DOTA)(H O)] (r2p = 4.57 m s ,
2
2
0 MHz). The r /r ratio for [Gd(DO3A-Pr-ATP)(H O) ]
2p 1p 2 2
is 1.16, showing that the complex is a T -weighted contrast
1
agent. The maximum signal intensity attained is determined
[
36]
co-workers
have reported that the formation of ternary
by the r /r ratio, with the highest signal intensity occur-
2p 1p
inclusion complexes between the hydrophobic cavity of β-
ring when the ratio is unity.
III
cyclodextrin and Gd chelates leads to a 10-fold increase
of r . Many complexes form inclusion complexes with β-
1
p
cyclodextrin through noncovalent interactions between the
Conclusions
substituent and β-cyclodextrin cavity.[ The nature of the
9]
[
13]
substituent also plays an important role. Aime et al. have
In this work, the synthesis and relaxivity studies of a
reported a large KD (dissociation constant) value as a con- GdIII complex of adenosine 5Ј-triphosphate conjugated
sequence of the poor fit of the substituent into the β-cyclo- DO3A has been reported. The increase in the molecular
dextrin cavity compared to complexes with no substituent. weight of the chelate leads to an increase in the r1p and r2p
The presence of the substituent in the para position of the relaxivities due to the slow tumbling rate of the complex in
phenyl ring is expected to strengthen the binding and favor solution. The covalent attachment of ATP causes the com-
the formation of the inclusion complex.[ Thus, the en- plex to remain intact under different experimental condi-
hancement of the relaxivity of [Gd(DO3A-Pr-ATP)(H O) ] tions and it is expected to be biocompatible and nontoxic.
37]
2
2
in the presence of β-cyclodextrin is attributed to the forma- [Gd(DO3A-Pr-ATP)(H O) ] is expected to reduce the in
2
2
tion of the inclusion complex by a noncovalent host–guest vivo clearance rate due to its noncovalent interaction with
interaction between the free amine group in the para posi- blood serum albumin and could be used as an intravascular
tion of the phenyl ring of ATP and the hydrophobic cavity contrast agent as the persistence of the complex in blood
of β-cyclodextrin.
A plot of the concentration of would be higher. Thus, there is a wide scope for studying
[
Gd(DO3A-Pr-ATP)(H O) ] vs. 1/T in the presence and the interaction and relaxivity of this complex in the pres-
2
2
1
absence of β-cyclodextrin is given in Figure 4.
ence of blood serum albumin to evaluate its suitability as a
blood pool agent. Furthermore, the ATP-conjugated deriv-
pH Dependence of the Relaxivity
ative is particularly interesting since the NH group pro-
2
The relaxivity of [Gd(DO3A-Pr-ATP)(H O) ] at pH 8.4 vides a site for further modification of the ligand or coval-
2
2
–
1 –1
(
TRIS buffer) is 5.64 m s , which is one unit lower than ent attachment of a substituent. The appended ATP does
the relaxivity of the complex without pH adjustment not bind with the central metal ion or block the coordina-
pH 5.4). The hydration number of the metal plays an im- tion site of the inner-sphere water molecules and remains
(
3
+
portant role on relaxivity. [Gd(H O) ] has a q value of 8 as an extended pendant arm. It is worthwhile to study the
or 9 and exhibits a relaxivity of 11.8 m s . As the q behavior of this complex in response to biological events
value decreases, the relaxivity also decreases. Sherry et al.
2
8
–
1 –1
[
38]
such as enzyme action. The advent of attaching metal com-
have observed that the decrease in relaxivity at higher pH plexes to larger biological molecules has created a new
Eur. J. Inorg. Chem. 2005, 3918–3927
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3923

S. J. Ratnakar, V. Alexander
FULL PAPER
realm of research in the field of bioconjugates. Conjugates relaxation time, T
1
. The T
1
measurements were carried out on a
Maran wide line NMR (Resonance Instruments Ltd., UK) op-
erating at 24 MHz and 35± 0.1 °C. The temperature was controlled
using a temperature console. The complex solutions in triply dis-
tilled water were placed in a 10-mm stoppered quartz tube and the
of chelates with biological macromolecules that have affin-
ity for specific tissues can be employed to study specific
biological processes and physiological function. Design of
MRI contrast agents that remain in the body for longer
time and target specific tissues is a need for the future.
1
instrument parameters were optimized for each T measurement.
The T measurements were made using the standard inversion re-
1
covery pulse sequence (180°–τ–90°) with phase-sensitive detec-
tion[ with varying τ values ranging from 50 µs to 6 s for each
concentration of the complex. The computer program Winfit was
used to plot the time vs. signal intensity to get an exponential plot
41]
Experimental Section
Chemicals: Triethylenetetraamine (99%), p-toluenesulfonyl chlo-
ride, 1,2-dibromoethane, 1,3-dibromopropane, chloroacetic acid,
xylenol orange, β-cyclodextrin, Celite, anhydrous potassium car-
bonate, and TRIS (Fluka) were used as such. Adenosine 5Ј-triphos-
phate disodium salt (Aldrich) was converted into the free form by
and the T
least 5T was maintained in between successive pulses to allow the
complete return of the spin system to equilibrium. The T values
for five different concentrations of the complex were measured. A
plot of 1/T vs. concentration of the complex gave a straight line
1
values were calculated from the plot. A delay of at
1
1
1
+
passing through Amberlite IR120 (H , strongly acidic) (Aldrich)
and the slope was taken as the longitudinal relaxivity r1p. The longi-
tudinal relaxivity at pH 8.5 was measured by maintaining the pH
by adding TRIS buffer. The longitudinal relaxivity in the presence
of β-cyclodextrin was determined by adding aqueous solution of a
known amount of β-cyclodextrin and keeping the solution over-
night at ambient temperature to attain equilibrium. Six different
concentrations (0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 m) of the complex
in water were used for relaxivity measurements.
cation exchange resin and eluting with triply distilled water. The
resin was washed with distilled water five times before use. Triethyl-
amine, perchloric acid (Merck, India), sodium hydroxide and po-
tassium hydroxide pellets (Rankem, India), and gadolinium() car-
bonate (Indian Rare Earths Ltd.) were used as such. Gd(ClO
4 3
)
·
nH O was prepared from gadolinium() carbonate and perchloric
2
acid in water. The perchlorate salt was recrystallized from triply
distilled water prior to use. Cyclen was synthesized by the method
Transverse Relaxivity (r2p) Measurements: The transverse relaxivity,
[
39]
of Chavez and Sherry. The solvents were purified by the standard
[
40]
2
r2p, was determined from the spin–spin relaxation time T . A stan-
procedures.
dard CPMG (Carl–Purcel–Meiboon–Gill) pulse sequence (90°–τ–
180°)[ with a τ value of 50 µs was used to determine T . The
42]
Physical Measurements: Infrared spectra were recorded on a Per-
kin–Elmer Spectrum RX-I FT-IR spectrometer in the range of
2
computer program Winfit was used to plot the time vs. signal inten-
sity to get an exponential curve and T2 was calculated from the
graph. The T2 values for six different concentrations were mea-
sured. The transverse relaxivity was calculated from the slope of
the regression line, obtained by the plot of the concentration of the
complex vs. 1/T2 by a least-squares fitting method. Six different
concentrations (0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 m) of the complex
were prepared in triply distilled water.
4
000–400 cm–1 using KBr pellets. Potassium bromide (FT IR grade,
Aldrich) was used to make pellets. FAB mass spectra were recorded
on a Jeol SX-102/DA 6000 mass spectrometer/data system using
argon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was
10 kV and the spectra were recorded at room temperature using
m-nitrobenzyl alcohol as the matrix. Electrospray ionization mass
spectra were recorded with a Micromass Quattro-II Triple Quatra-
pole mass spectrometer. The sample was dissolved in water and
introduced into the ESI capillary using a 5-µL syringe pump. The
ESI capillary was set at 3.5 kV with a cone voltage of 40 V. CHN
microanalyses were carried out using Perkin–Elmer 2400 Series II
CHNS/O Elemental Analyzer interfaced with a Perkin–Elmer AD
1,4,7-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A)
(
2): Chloroacetic acid (2.820 g, 30 mmol) in 20 mL of water was
placed in a double-walled, round-bottomed flask, connected to a
cryostat maintained at –4 °C, and aqueous sodium hydroxide (5 )
was added dropwise whilst stirring until the pH reached 5. Cyclen
sulfate (3.684 g, 10 mmol) in water (50 mL) was neutralized with
6
Autobalance. Helium (analytical grade) was used as the carrier
gas. Analytical and preparative HPLC analysis were carried out
using Varian PrepStar 218 (Varian Instruments Inc., USA) binary
gradient solvent delivery module with an inline three channel de-
gasser Model 2000 for solvent delivery. A Rheodyne injector valve
5
 sodium hydroxide and added to the chloroacetic acid solution
in the flask whilst stirring at –4 °C. The pH of the mixture was
increased to 10 by the addition of 5  aqueous sodium hydroxide.
The solution was maintained at –4 °C with constant stirring whilst
maintaining pH 10 by the frequent addition of 5  aqueous sodium
hydroxide for 3 h. The contents of the flask were transferred to
another round-bottomed flask, fitted with a double surface con-
denser, and slowly heated to room temperature for 5 h while main-
taining a pH of 10. The solution was further heated to 50 °C over
a period of 4 h and the temperature was maintained for 8 h at
pH 10. The solution was cooled to room temperature and loaded
(
(
20 µL) was used for the sample injection. HPLC column
250×4.6 mm×1/4 in Valco, Microsorb-MV 100-5 C18) (analyti-
cal) and DYNAMAX HPLC column (250×10.0 mm, Microsorb
3
3
00–10 C18) (preparative) were used. UV/Visible detector Model
45 operating in the range 190–1100 nm was used. The fractions
were collected using a Model 704 fraction collector. EPR spectra
were recorded on a Jeol instrument at Q-band (34.5 MHz) and X-
band (9.4 MHz) with a scan range of 8000 g and the field was set
at 12500 T. Magnetic susceptibility measurements were carried out
+
onto a column containing Amberlite IR 120 (H , strongly acidic)
cation exchange resin and eluted with deionized water. The solvent
was removed under reduced pressure in a rotary evaporator and
the resulting white solid was washed with dry acetone and meth-
anol, filtered, and dried in a vacuum desiccator. The purity of
on EG&G PAR Model 155 vibrating sample magnetometer at
1
2
5 °C. H NMR spectra were recorded in D
2
O and CDCl
3
(9.995
atom% D, Aldrich) on a Jeol GSX-400 multinuclear NMR spec-
trometer working at 400 MHz at 25 °C. The 13C NMR spectra were
DO3A was checked by HPLC (R
yield 69.58%; m.p. 260 °C. IR (KBr): ν˜ = 3406.5 cm
964.0 ν (CH), 1723.0 ν (C=O), 1623.6 δ(NH), 1459.8 ν(CH),
1391.6 δ(OH), 1212.3 ν (CO), 1087.0 ν (CN), 985 δ(CH), 720
(CH). MS (70 eV, EI): m/z (%) = 344 (80) [M – 2] (C14
f 2 3
= 3.244, H O/CH CN 9:1 v/v);
3
1
recorded at 100 MHz using a Jeol GSX-400 instrument. The
NMR spectra were recorded at 162 MHz.
P
–
1
s
ν (OH),
2
s
s
Longitudinal Relaxivity (r1p) Measurements: The longitudinal re-
s
s
III
+
laxivity of the Gd complex was determined from the spin-lattice
δ
r
24 4 6
H N O ),
3924
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
Eur. J. Inorg. Chem. 2005, 3918–3927

Gadolinium() Complex as a Contrast Enhancing Agent for MRI
FULL PAPER
3
29 (50) [M
–
NH]+ (C14
H
23
N
3
O
6
), 302 [M
–
C
2
NH
3
]+
[Gd(DO3A-Pr-ATP)(H
2
O)
2
] (4): Gadolinium() perchlorate hy-
+
(
C
12
H
20
N
3
O
6
), 243 (20) [M – C
4
H
7
NO
), 57 (100) (C
O, TMS): δ = 3.21 (m, 12 H, CH
), 3.60 (s, 6 H, CH COOH), 3.9 (d, 1 H, J = 5.3 Hz, NH)
O): δ = 42.5 (CH ), 47.8 (N– perature and the absence of uncomplexed Gd ions was confirmed
COOH), 174.5 (COOH) ppm. C14
346.08): calcd. C 48.58, H 7.51, N 16.18; found C 48.14, H 7.43,
N 15.94.
2
]
(C10
H
H O
2 2 2
17
N
3
O
4
), 185 (50) drate (0.11 g, 0.25 mmol) was added to a solution of DO3A-Pr-
). 1H NMR ATP (0.22 g, 0.25 mmol) in 30 mL of water and stirred at room
), 3.39 (m, 4 H, temperature for 36 h. Dowex cation exchange resin (10 g) was
added to the reaction mixture and stirred for 15 min at room tem-
+
[M – C
6
H
9
NO
4
]
(C
8
H
15
N
3
O
2
(400 MHz, D
2
2
N–CH
2
2
ppm. 13C NMR (100.34 MHz, D
III
2
2
by testing the solution with xylenol orange indicator.[ The solu-
tion was filtered to remove solid particles and the filtrate was dis-
tilled off under reduced pressure in a rotary evaporator to dryness.
The white solid was recrystallized from triply distilled water. The
purity of the complex was checked by HPLC (single peak, retention
43]
CH
(
2
), 52.06 (CH
2
26 4 6
H N O
10-Bromopropyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-
dodecane (3): DMF (30 mL) and 5 drops of triethylamine were
added, whilst stirring, to a solution of DO3A (3.45 g, 10 mmol) in
time 4.420 (H
IR (KBr): ν˜ = 3303.4 cm
238.0 ν (P=O), 909 ν (P–O). MS (ESI): m/z = 1081 [M – 2]
Gd(C27 )(H
)], 734 [M – C12
2 3
O/CH CN, 9:1, v/v); yield 59.09%; m.p. 175 °C (dec).
–
1
s s
ν (OH), 1608.8 (COO), 1410.6 δ (OH),
60 mL of distilled water in a round-bottomed flask, kept over a
+
1
s
s
rotamantle, and the mixture was heated to 80 °C. The temperature
was maintained at 80 °C for 1 h with stirring. 1,3-Dibromopropane
+
[
H
41
N
9
O
19
P
3
2
O)
2
], 793 [M – C11
H
17
N
5
O
5
] [Gd(C17
)], 578
), 483 [M – Gd(C12
), 168 [M – Gd(C19
)(H O) (1083.05): calcd. C 29.94,
27
H -
+
N
4
O
16
P
3
H
21
N
5
O
7
]
24 4 14 3
[Gd(C15H N O P
(2.01 g, 10 mmol) in 10 mL of DMF was then added slowly and
+
[
N
M – Gd(C12
H
20
N
5
O
N
7
)] (C15
H
25
N
4
O
14
P
3
20
H -
the reaction mixture was stirred for 36 h at 80 °C. The mixture was
filtered and the filtrate was evaporated in a rotary evaporator to
dryness. The solid compound that separated out was washed with
dry methanol to remove triethylamine hydrobromide salt and then
with acetone. The white solid was dried in a vacuum desiccator and
recrystallized from water. The purity of 3 was checked by HPLC
+
29 5 21 3
H N O P
)]+
5
O11P)] (C15
H
25
4
O
10
P
2
(
C
8
H
16
N
4
). Gd(C27
H
43
N
9
O
19
P
3
2
2
H 4.33, N 11.63; found C 29.68, H 4.25, N 11.46.
Supporting Information Available (see footnote on the first page of
this article): Chart S1. Gadolinium() chelates approved by FDA
for human imaging. FAB mass spectroscopic data of 10-(3-bro-
mopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-
(R
f
= 4.20, H
IR (KBr): ν˜ = 3432.2 cm
460.3 δ(CH), 1385.4 δ(OH), 1214.4 ν
δ(CH), 699.4 (CH). MS (FAB): m/z
Br), 341 [M – C
2 3
O/CH CN, 3:2 v/v); yield 67%; m.p. 290 °C (dec).
–
1
ν
s
(OH), 2954.0 ν
(CO), 1087.3 ν
466 [M
), 284 [M –
Br] (C10 ).
O, TMS): δ = 2.63 (m, 2 H, CH ), 3.23
s
(CH),1692.0 ν
s
(C=O),
dodecane (Figure S1), T
1
values for different concentrations of
1
s
s
(CN), 980.7
+
[Gd(L1)(H O) ] (Table S1), the plot of concentration of
δ
r
=
–
1]
2
2
+
2 2 1 1p
[Gd(L1)(H O) ] vs. 1/T (r relaxivity measurements) (Figure S2),
T1 values for different concentrations of [Gd(L1)(H O) ] in the
2 2
(C
17
H
30
N
4
O
6
+
3
H
9
Br] (C14
H
21
N
4
+
O
6
C
5
H
11
O
2
Br] (C12
H NMR (400 MHz, D
m, 12 H, CH ), 3.41 (s, 6 H, N–CH
NMR (100.34 MHz, D O): δ = 36.84 (N-CH
8.28 (CH COOH) 174.31 (COOH) ppm. C17
calcd. C 43.71, H 6.63, N 11.99; found C 43.18, H 6.33, N 11.53.
H
20
N
4
O
4
), 255 [M – C
7
H
16
O
2
H
15
N
4
O
4
1
presence of β-cyclodextrin (Table S2), the plot of concentration of
[Gd(L1)(H O) ] vs. 1/T in the presence of β-cyclodextrin (Fig-
2
2
1
3
(
2
2
), 3.84 (CH
2
COOH) ppm.
C
2
2
1
ure S3), T
at pH 8.4 (Table S3), and a plot of concentration of [Gd(L1)-
O) ] vs. 1/T (r1p relaxivity measurements at pH 8.4; Figure S4).
1 2 2
values for different concentrations of [Gd(L1)(H O) ]
2
2
-CH
2
), 52.73 (CH
2
),
5
2
H31BrN O (467.01):
4 6
(
H
2
2
1
1
0-[3-(Adenosine5Ј-triphosphate)oxypropyl]-1,4,7-tris(carboxyme-
thyl)-1,4,7,10-tetraazacyclododecane (DO3A-Pr-ATP): Adenosine Acknowledgments
5Ј-triphosphate disodium salt (0.551 g, 1 mmol) was dissolved in
This research was carried out with the financial support of the
Board of Research in Nuclear Sciences (BRNS), Department of
Atomic Energy, Government of India. We thank Dr. V. K. Manch-
anda, Head, Radiochemistry Division, Bhabha Atomic Research
Center, Mumbai-400085, for his support. We thank Dr. Venkat
Manohar, Measurements Science Division, Unilever Research In-
dia, Bangalore, for providing facilities for relaxivity measurements.
5
mL of water and passed through a column packed with Amberlite
+
IR-120 (H , strongly acidic) cation exchange resin and eluted with
distilled water. The linker-appended DO3A (3; 0.466 g, 1 mmol) in
25 mL of water was added to the eluent, which contains free ATP,
and stirred for 24 h at room temperature. The solvent was removed
under reduced pressure in a rotary evaporator whilst maintaining
the bath temperature below 40 °C. The vitreous solid that separated
out became white upon the addition of methanol and was washed
with acetone, dried in a vacuum desiccator, and stored in a refriger-
[1] The 2003 Nobel Prize in Physiology or Medicine, The Nobel
Assembly, Karolinska Institutet, Stockholm, Sweden, October
ator. The purity of DO3A-Pr-ATP was checked by HPLC (R
.11, H O/CH CN, 9:5 v/v); yield 53.4%; m.p. 153 °C (dec). IR
KBr): ν˜ = 3420.1 cm
700 ν (C=O), 1635.4 δ
δ(C–H), 719.8 δ (C–H), 1228.4 ν
m/z = 894 [M + 1] (C27
f
=
6, 2003.
6
(
1
2
3
–1
[2] a) P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem.
Rev. 1999, 99, 2293–2352; b) M. F. Tweedle, in Lanthanide
Probes in Life, Chemical, and Earth Sciences (Eds.: J.-C. G.
Bunzli and G. R. Choppin), Elsevier, Amsterdam, 1989; c)
R. B. Lauffer, Chem. Rev. 1987, 87, 901–907; d) K. Kumar,
M. F. Tweedle, Pure Appl. Chem. 1993, 65, 515–520; e) A. E.
Merbach, E. Toth, The Chemistry of Contrast Agents in Medi-
cal Magnetic Resonance Imaging, Wiley-VCH, Weinheim, 2001;
f) J. A. Peters, J. Huskens, D. J. Raber, Prog. Nucl. Magn. Re-
son. Spectrosc. 1996, 28, 283–350.
ν
s
(OH), 2926.5 ν
(NH), 1507.1 ν (C–O), 1087.6 ν
(P=O), 900 ν (P–O). MS (ESI):
), 756 [M – C
s
(CH), 1476.8 δ
s
(CH),
s
s
s
s
(C–N), 995
r
s
s
+
+
H
47
N
9
O
19
P
3
5 7 5
H N ]
+
(C
22
H
39
N
H
4
O
19
P
3
), 654 [M – C
9
H
13
N
5
O
3
]
(C18
H
33
N
4
O
16
P
3
), 348
). H NMR (400 MHz, D O,
-CH ), 3.10–3.4 (m, 12 H,
), 4.25 (t, 1 H),
COO), 4.49–4.65 (d, J = 7.08 Hz, 2 H), 6.073 (m, 1
+
1
[M – C13
19
N
5
O
13
P
3
]
(C14
H
28
N
4
O
6
2
TMS): δ = 2.25 (t, 2 H, N-CH
NCH CH
.37 (6 H, CH
H), 8.37, 8.54 (m, 2 H) ppm. C NMR (100.34 MHz, D
4.78 (CH ), 52.99 (CH ), 57.23 (CH COOH), 68.43 (OCH
OCH ), 87.14 (C , C , and C of furan ring), 174.52 (COOH),
52.90–145.57 (aromatic ring) ppm. P NMR (162 MHz, D
2
2
2
2 2
N), 3.58–3.63 (t, J = 5.5 Hz, 2 H; OCH
4
2
13
[3] P. C. Lauterbur, M. H. Mendonca-Dias, A. M. Rudin, in Fron-
tiers of Biological Energetics (Eds.: P. L. Dutton, L. S. Leigh,
A. Scarpa), Academic Press, New York, 1978.
2
O): δ =
2
), 77.38
3
(
1
=
C
2
2
2
2
2
3
4
[
4] M. F. Tweedle, G. T. Gaughan, J. Hagan, P. W. Wedeking, P.
31
2
O): δ
107.35 (P–P–P), 119.30 (P–P–P), 130.20 (P–P–P) ppm.
(893.05): calcd. C 36.31, H 5.15, N 14.10; found C
Sibley, L. J. Wilson, D. W. Lee, Nucl. Med. Biol. 1988, 15, 31–
36.
27
H
46
N
9
O
19
P
3
[5] a) I. Bertini, C. Luchinat, NMR of Paramagnetic Molecules in
Biological Systems, Benjamin/Cummings Publishing Co.,
36.14, H 4.96, N 13.94.
Eur. J. Inorg. Chem. 2005, 3918–3927
www.eurjic.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3925

S. J. Ratnakar, V. Alexander
FULL PAPER
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