Thermodynamic stability and relaxation studies of small, triaza-macrocyclic Mn(ii) chelates

Article abstract of DOI:10.1039/c2dt32496b

Due to its favorable relaxometric properties, Mn²⁺ is an appealing metal ion for magnetic resonance imaging (MRI) contrast agents. This paper reports the synthesis and characterization of three new triazadicarboxylate-type ligands and their Mn<

Full text of DOI:10.1039/c2dt32496b

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Thermodynamic stability and relaxation studies of
small, triaza-macrocyclic Mn(II) chelates†
Cite this: Dalton Trans., 2013, 42, 4522
Arsénio de Sá,a Célia S. Bonnet,b Carlos F. G. C. Geraldes,c Éva Tóth,b
Paula M. T. Ferreira*a and João P. André*a
Due to its favorable relaxometric properties, Mn2+ is an appealing metal ion for magnetic resonance
imaging (MRI) contrast agents. This paper reports the synthesis and characterization of three new triaza-
dicarboxylate-type ligands and their Mn2+ chelates (NODAHep, 1,4,7-triazacyclononane-1,4-diacetate-
7-heptanil; NODABA, 1,4,7-triazacyclononane-1,4-diacetate-7-benzoic acid; and NODAHA, 1,4,7-triaza-
cyclononane-1,4-diacetate-7-hexanoic acid). The protonation constants of the ligands and the stability
constants of the chelates formed with Mn2+ and the endogenous Zn2+ ion have been determined by
potentiometry. In overall, the thermodynamic stability of the chelates is lower than that of the corres-
ponding NOTA analogues (NOTA
= 1,4,7-triazacyclononane-1,4,7-triacetate), consistent with the
decreased number of coordinating carboxylate groups. Variable temperature 1H NMRD and 17O NMR
measurements have been performed on the paramagnetic chelates to provide information on the water
exchange rates and the rotational dynamics. The values of the 17O chemical shifts are consistent with the
presence of one water molecule in the first coordination sphere of Mn2+. The three complexes are in the
slow to intermediate regime for the water exchange rate, and they all display relatively high rotational
correlation times, which explain the relaxivity values between 4.7 and 5.8 mM−1 s−1 (20 MHz and 298 K).
These relaxivities are higher than expected for Mn2+ chelates of such size and comparable to those of
small monohydrated Gd3+ complexes. The amphiphilic [Mn(NODAHep)] forms micelles above 22 mM
(its critical micellar concentration was determined by relaxometry and fluorescence), and interacts with
HSA via its alkylic carbon chain providing a 60% relaxivity increase at 20 MHz due to a longer tumbling
time.
Received 19th October 2012,
Accepted 21st December 2012
DOI: 10.1039/c2dt32496b
www.rsc.org/dalton
Introduction
Magnetic resonance imaging (MRI) has become one of the
most successful diagnostic imaging modalities of the last few
aCentro de Química, Campus de Gualtar, Universidade do Minho, 4710-057 Braga,
Portugal. E-mail: jandre@quimica.uminho.pt; Fax: +351 253 604 382;
Tel: +351 253 604 385
decades. A large number of MRI scans are based on the use of
paramagnetic contrast agents (CAs) which enhance the con-
trast of images of organs and tissues. Paramagnetic metal ions
such as Gd3+ and Mn2+ are adequate for the preparation of CAs
due to their high magnetic moment, long electronic relaxation
times and the lability of their coordinated water molecules.
MRI contrast agents act by increasing both longitudinal (1/T1)
and transverse (1/T2) relaxation rates of water protons in the
human body, and their efficiency is expressed by their water
proton relaxivity, ri (i = 1, 2).1
bCentre de Biophysique Moléculaire, CNRS, rue Charles Sadron, 45071 Orléans,
France
cDepartamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Centro de
Neurociências e Biologia Celular and Centro de Química de Coimbra, Universidade
de Coimbra, 3001-401 Coimbra, Portugal
†Electronic supplementary information (ESI) available: Fig. S1. Determination of
cmc of [Mn(NODAHep)] by paramagnetic relaxation enhancement measure-
ments. Fig. S2. Potentiometric titration curves of solutions containing NODAHA
1.34 mM with 0 or 1 equiv. of Mn2+ or Zn2+ in H2O, KCl 0.1 M, 298 K. Fig. S3.
Potentiometric titration curves of solutions containing NODABA 1.55 mM with 0
or 1 equiv. of Mn2+ or Zn2+ in H2O, KCl 0.1 M, 298 K. Fig. S4. (Top) Temperature
dependence of the reduced 17O transverse relaxation rate of [Mn(NODAHA)].
(Bottom) 1H NMRD profiles of [Mn(NODAHA)] at 298, 310 and 323 K. Fig. S5.
(Top) Temperature dependence of the reduced 17O transverse relaxation rate of
[Mn(NODABA)]. (Bottom) 1H NMRD profiles of [Mn(NODABA)] at 298, 310 and
323 K. Fig. S6. 1H NMRD profile of [Mn(NODAHep)] in the presence of HSA at
298 K. Equations used for the analysis of NMRD and 17O NMR data. See DOI:
10.1039/c2dt32496b
Thanks to its seven unpaired electrons and long electronic
relaxation time, Gd3+ is the most widely used paramagnetic
metal ion in MRI. To prevent toxicity, Gd3+ has to be adminis-
tered to the patient in the form of a thermodynamically and
kinetically stable chelate. Several gadolinium-based chelates
have been approved as CAs, including Magnevist®
([Gd(DTPA)]−, DTPA
=
diethylenetriaminepentaacetate)2 or
4522 | Dalton Trans., 2013, 42, 4522–4532
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atoms, of which one could be in a pyridine moiety (py), e.g. 15-
aneN5,20 Me2-15-pydieneN5,21 15-pyN5 or 15-pyN3O2.22
In the development of novel and better MRI contrast
agents, two main goals are commonly considered: higher relax-
ivity and specificity for organs or tissues. Human serum
albumin (HSA), the most abundant blood serum protein, pre-
senting high affinity for long carbon chain molecules,23 is an
interesting biological target for CAs, especially those aimed at
acting as blood pool agents. Chelates bearing hydrophobic
moieties can bind to HSA, thus increasing their blood reten-
tion time and, simultaneously, increasing their relaxivity due
to longer tumbling times.9,24–26
Several strategies have been suggested for the delivery of
drugs, among them self-aggregating molecules that can form
micelles.27–29 This delivery route is known to be promoted by
macrophage-rich tissues such as liver and spleen.30 Micelliza-
tion of amphiphilic paramagnetic chelates has the additional
advantage of increasing the relaxivity thanks to an increase in
the tumbling time of the chelate in solution.31–36 Another
strategy for the development of CAs targeted for specific
tissues and organs consists in their coupling to biologically
relevant molecules (monoclonal antibodies, peptides, peptide
Scheme 1 Chemical structure of various chelators for Mn2+ discussed in this
work.
Dotarem® ([Gd(DOTA)]−, DOTA = 1,4,7,10-tetraazacyclodode-
cane-1,4,7,10-tetraacetate).3
mimetics or non-peptidic substrates and carbohydrates37–42
that are recognized by specific receptors.
)
Despite its favorable features (five unpaired d electrons,
long electronic relaxation times and labile coordinated water
molecule(s)), Mn2+ received less attention and there has been
only one approved Mn2+-based contrast agent (Teslascan®,
[MnDPDP]4−, DPDP6− = N,N′-dipyridoxylethylenediamine-N,N′-
diacetate-5,5′-bis-(phosphate), Scheme 1).4,5 A renewed interest
in Mn2+-based MRI CAs has led to a systematic development
and study of Mn2+ chelates.6 These include linear polyamino-
In this paper, we report the synthesis of three new triaza-
polycarboxylate-based ligands for Mn2+, NODAHep (1,4,7-tria-
zacyclononane-1,4-diacetate-7-heptanil),
triazacyclononane-1,4-diacetate-7-benzoic acid), and NODAHA
(1,4,7-triazacyclononane-1,4-diacetate-7-hexanoic acid)
NODABA
(1,4,7-
(Scheme 1) and the solution characterization of the stability
and relaxometric properties of their Mn2+ complexes using
potentiometric and relaxometric NMR techniques. Being penta-
dentate, these ligands maintain a coordination site of the
metal ion available for one water molecule. NODAHep has a
lipophilic side chain which was introduced to endow its che-
lates with the capability of forming micelles in solution and,
additionally, to interact non-covalently with blood serum pro-
teins, in particular HSA. NODABA and NODAHA were designed
to act as bifunctional ligands which can be conjugated to tar-
geting molecules.
carboxylates like EDTA7,8 (EDTA = ethylenediamine tetraacetic
9
acid) and its derivatives such as EDTA-BOM1,2
and
diphEDTA10 (bearing one or two BOM = benzyloxymethyl or a
diph = diphenylcyclohexylphosphate substituent(s), respect-
ively), or derivatives of DTPA10–13 (DTPA = diethylenetriamine
pentaacetic acid). In these EDTA-type complexes, the Mn2+ ion
has a coordination number (CN) of 7 with one inner sphere
coordinated water molecule. Chelates of Mn2+ with macro-
cyclic polyamino-polycarboxylate ligands which have been
described include derivatives of triazacyclononane (NOTA14
(NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid) and the
dimeric ENOTA15), of diaza-oxacyclononane (9-aneN2O deriv-
atives16), of tetraazacyclododecane (DOTA14) and its derivatives
bearing four or less pendant arms,9,17,18 such as DOTAM
(DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceta-
mide), (DO3A(BOM)3 (DO3A = 1,4,7,10-tetraazacyclododecane-
1,4,7-triacetic acid, BOM = benzyloxymethyl) and DO2A =
1,4,7,10-tetraazacyclododecane-1,7-diacetic acid), and a 1,4-di-
azepine-based ligand (AAZTA = 6-amino-6-methylperhydro-1,4-
diazepine triacetic acid).19 The Mn2+ complexes of these
ligands have a maximum of one water molecule on the first
coordination sphere, and the DOTA, DOTAM and DO3A(BOM)3
complexes have no inner-sphere water. Bishydrated Mn2+ com-
plexes have been reported with ligands based on 15- or larger
membered aza- or aza-oxa crown ethers containing five donor
Results and discussion
Synthesis
The synthetic strategy used for the preparation of NODAHep,
NODABA and NODAHA involved the alkylation of 1,4,7-triaza-
cyclononane-1,4-diacetic acid tert-butyl ester (NO2AtBu) fol-
lowed by removal of the protecting groups (Scheme 2). In the
case of NODAHep, 1-bromoheptane was reacted with NO2AtBu
in the presence of potassium carbonate to give NODA(tBu)-
Hep. This compound was then treated with trifluoroacetic acid
(TFA) to give NODAHep in a 78% yield. The synthesis of
NODABA and NODAHA was carried out using as alkylating
agents the 9-fluorenylmethyl ester of 4-(bromomethyl)benzoic
acid and the methyl ester of 6-bromohexanoic acid to give
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Scheme 2 (a) K2CO3 in MeCN; (b) TFA in DCM; (b’) 1 M NaOH in MeOH, Δ; (c) MnCl2·4H2O in 0.5 M HEPES buffer, pH 7.4.
NODA(tBu)BA and NODA(tBu)HA(me) in high yields. The
NODABA and NODAHA were obtained by treating NODA(tBu)-
BA with trifluoroacetic acid and NODA(tBu)HA(me) with a solu-
tion of NaOH. The unprotected carboxylic acid present in
NODABA and NODAHA can be used to link a vectorizing
moiety.
The preparation of the Mn(II) chelates was done in HEPES
buffer, pH 7.4. The formation of the chelates occurred rapidly.
Determination of the critical micelle concentration
The critical micelle concentration (cmc) of [Mn(NODAHep)]
was determined by two methods. In the first method a fluo-
rescence probe was used, which was 8-anilino-1-naphthalene
sulfonic acid (ANS), as its luminescence is sensitive to the
Fig. 1 Determination of cmc by fluorescence spectroscopy. The intersection of
the linear regression curves of the fluorescence intensity of ANS at 480 nm as a
function of the chelate concentration determines the cmc of NODAHep.
medium polarity. In polar environments, such as in water,
ANS is essentially non-fluorescent and in non-polar environ-
ments, such as the inner part of micelles, it is highly
fluorescent.43–45 In order to evaluate the micelles formation,
the fluorescence intensity of ANS was measured at 480 nm in
the presence of different chelate concentrations. As the
micelles are forming, ANS is entrapped in the micellar com-
partment and an increase of its fluorescence can be
detected.44,45 A cmc value of 22.3 mM was determined by
linear least-squares fitting of the fluorescence emission at
480 nm versus the concentration of the [Mn(NODAHep)]
chelate (Fig. 1). The alternative method consisted in the
measurement of the longitudinal relaxation rate of the water
hydrogen nuclei with increasing concentrations of [Mn-
(NODAHep)], at 40 MHz and 298 K. Indeed, at concentrations
below the cmc, no micelles are formed, and therefore the
proton relaxation rate measured in the solution is only due to
the free, non-aggregated paramagnetic complex and can be
expressed as follows:
the Mn2+ concentration. Above the cmc, the measured relax-
ation rate is the sum of two contributions, one from the non-
aggregated complex present at a concentration equal to the
cmc, and one of the complex in the micellar form. The para-
magnetic relaxation enhancement in this case is given by:
R1 ¼ rn1a ꢀ cmc þ r1aðCMn ꢁ cmcÞ ¼ ðr1na ꢁ ra1Þcmc þ r1aCMn ð2Þ
where ra1 is the relaxivity of the aggregated form of the
complex.
The cmc is then determined from the plot of R1 as a func-
tion of CMn, and by a simultaneous least-squares fit of the two
straight lines. The intercept of those two lines gives the value
of the cmc, 22.8 mM in this case (Fig. S1†), very much consist-
ent with the result obtained in the previous method. It should
be noted that the relaxivity of the non-aggregated form is
R1 ¼ rn1aCMn
ð1Þ
1
found to be 4.51 mM−1 s−1 in accordance with the H NMRD
where R1 is the paramagnetic relaxation enhancement, rn1a is measurements (vide infra), and the relaxivity of the micellar
the relaxivity of the non-aggregated Mn2+ complex, and CMn is form is found to be 9.57 mM−1 s−1. This higher value of
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relaxivity was predictable, as in the micellar form the
rotational correlation time is expected to be bigger than in the
monomeric form.
Table 1 Protonation constants of various ligands at 298 K and in KCl = 0.1 M
NODAHep
NODAHA
NODABA
NOTA
The cmc values obtained for [Mn(NODAHep)] are higher
than those found for complexes such as [Ga(NOTAC8)] and [Al-
(NOTAC8)], 0.36 mM and 0.25 mM respectively (NOTAC8 =
Log KH1
Log KH2
Log KH3
Log KH4
Log KH5
12.0(3)
5.81(2)
2.71(8)
11.1(2)
6.00(5)
4.53(2)
2.74(5)
11.2(1)
5.35(3)
4.07(6)
3.04(9)
2.0(1)
11.41
5.74
3.16
1.71
1,4,7-triazacyclononane-1,4-diacetic
acid-7-octanoic
acid;
Scheme 1).46 This is somewhat surprising, considering that
NODAHep and NOTAC8 have the same length of alkyl chains,
and the three complexes have no charge. Given the high value
of cmc obtained for this complex, we will study only the mono-
meric form of the complex in the following.
been shown that the first two protonations of the ligand NOTA
occur on the ring nitrogen atoms, while the third protonation
occurs on a carboxylate oxygen atom.47 So for all the ligands,
we attribute the first two protonation constants to two nitrogen
atoms of the macrocycle, and the other protonation constants
are attributed to carboxylate functions. NODAHep is very
similar to “half” ENOTA (Scheme 1), which displays two macro-
cyclic units. And indeed, the protonation constants of
NODAHep are very close to those obtained for ENOTA (12.5,
12.2, 5.97, 5.18, 2.73, 1.86).15 For NODAHA, the third proton-
ation constant can be attributed to the carboxylate function on
the alkyl chain, as the value is close to the one of an isolated
carboxylate function. For NODABA, the second protonation on
the nitrogen is lower than the one of the other ligand, which
can be explained by the electron withdrawing effect of the
benzene ring. The third protonation constant can be attributed
to the benzoate function, as the pKa of the benzoic acid substi-
tuted in the para position by a methyl is 4.37.48
Determination of protonation and stability constants
The protonation constants, log KH , of the three ligands
i
NODAHep, NODAHA and NODABA, as defined in eqn (3), were
determined by pH-potentiometric titrations at I = 0.1 M KCl
and 298 K.
½HiLꢂ
KH
¼
ð3Þ
i
½Hiꢁ1Lꢂ½Hþꢂ
Three protonation constants could be determined for
NODAHep, four for NODAHA and five for NODABA. The titra-
tion curves are presented in Fig. 2 and S2, S3 (ESI†) and the
calculated protonation constants are shown in Table 1.
The potentiometric determination of protonation constants
1
Complex stability constants, log KML, complex protonation
constants, log KMLH, and hydroxo-complexes formation (eqn
(4)–(6)) have been determined for complexes formed with
Mn2+ and Zn2+ ions by direct potentiometric titrations, as the
formation of the complexes was fast.
above pH 11 is difficult, and they can be assessed by H NMR
spectroscopy if the chemical shifts of the non-labile protons of
the ligand exhibit a chemical shift change upon deproton-
ation. However in our case, attempts to determine these proto-
nation constants by NMR were unsuccessful as the chemical
shift changes observed were of the same order of magnitude
as the line broadening of the observed peaks. For this reason
the values obtained from potentiometric titrations, with a
large error taking into account the difficulty of their determi-
nation, have been considered.
½MLꢂ
KML
¼
¼
ð4Þ
ð5Þ
ð6Þ
½Mꢂ½Lꢂ
½MLHꢂ
KMLH
½Hꢂ½MLꢂ
The protonation sequence has been previously determined
for various cyclic polyaza polycarboxylate ligands, and it has
½MLꢂ
KMLðOHÞ
¼
½MLðOHÞꢂ½Hꢂ
The titration curves obtained at a 1 : 1 metal-to-ligand ratio
are presented in Fig. 2 and S2, S3 (ESI†). The stability con-
stants obtained from the fitting of the experimental curves for
the different complexes are summarized in Table 2.
The formation of a monoprotonated complex and a hydroxo-
complex has to be taken into account for NODAHA. The stab-
ility constants obtained for the Mn2+ complexes are similar for
the three ligands and they are 5–6 orders of magnitude lower
than the one obtained with Zn2+. In order to compare the ther-
modynamic stability of these complexes with those from the
literature, we calculated the pMn values for conditions com-
monly used for Gd3+ chelates (pH = 7.4, [Mn2+] = 10−6 M, [L] =
10−5 M). The values are similar for the three complexes, and
they are also similar to the one found for the binuclear
ENOTA complex (pM = 7.7).15 They are also in the same order
Fig. 2 Potentiometric titration curves of solutions containing NODAHep
3.08 mM with 0 or 1 equiv. of Mn2+ or Zn2+ in H2O, KCl 0.1 M, 298 K.
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Table 2 Stability constants, and pM values of NODAHep, NODAHA,
NODABA, and NOTA complexes with Mn2+ and Zn2+ ions at 298 K, and in KCl
0.1 M
Log K
NODAHep
NODAHA
NODABA
NOTAa
MnL
10.98(3)
10.15(5)
4.95(5)
9.65(5)
15.65(7)
4.76(2)
9.9(1)
14.9
MnLH
MnLOH
ZnL
16.6(1)
15.72(9)
4.25(8)
2.52(7)
1.26
18.6
ZnLH
ZnLH2
% Free Mn2+ b
pMnc
0.92
7.35
0.85
7.41
7.8 × 10−3
11.8
7.09
a From ref. 48 and 49. b Obtained at pH = 7.4 for [Mn] = [L] = 5 mM.
c pMn = −log[Mn]free for pH = 7.4, [Mn] = 10−6 M, [L] = 10−5 M.
of magnitude as the one found for pyridine-containing macro-
cyclic ligands with carboxylate, or phosphate as pendant
arms,22,50 or the ones of 9-aneN2O16 ligands with similar
pendant arms. They however remain 4–5 orders of magnitude
lower than those of MnNOTA, or MnEDTA, and 7 orders of
magnitude lower than the pMn of DOTA.48
17O NMR and NMRD measurements
In order to determine the microscopic parameters that rule
the relaxivity of the complexes, their 1H NMRD profiles have
been measured. These profiles represent the magnetic field
dependence of relaxivity on the proton Larmor precession fre-
quency and can be fitted by the Solomon–Bloembergen–
Morgan (SBM) theory. As a large number of microscopic para-
meters influence the 1H NMRD profiles, some of them need to
be determined by independent techniques such as 17O NMR.
The variable-temperature 17O transverse relaxation rates (1/T2)
give access to the exchange rate of the Mn2+-coordinated water
molecule, kex; the longitudinal relaxation rates (1/T1) provide
information about the rotational motion of the complex, and
hence τR, and finally the 17O chemical shifts (ω) inform about
the hydration number (q) of the complex.
Fig. 3 (Top) Temperature dependence of the reduced 17O transverse relaxation
rate of [Mn(NODAHep)]. (Bottom) 1H NMRD profiles of [Mn(NODAHep)] at
■
▲
●
298 K ( ), 310 K ( ), and 323 K ( ).
temperature, 1/T2r is defined by the transverse relaxation rate
of the bound water oxygen, 1/T2m, which in turn is influenced
by the water exchange rate, kex, the longitudinal electronic
relaxation rate, 1/Tle, and the scalar coupling constant, A/ħ.
The transverse 17O relaxation is governed by the scalar relax-
ation mechanism and thus contains no information on the
rotational motion of the system. If we are not interested in
detailed information about the electron spin relaxation and if
we restrict the analysis of the NMRD data to medium and high
magnetic fields, the SBM approach gives reliable information
on dynamic processes like water exchange and rotational corre-
lation times for small complexes.51 Therefore only relaxivity
values above 6 MHz have been included in the simultaneous
fit and the following parameters have been thus adjusted: the
water exchange rate, kex298, the activation enthalpy for water
exchange, ΔH≠, the scalar coupling constant, A/ħ, the
rotational correlation time, τR298, and its activation energy, ER,
and the parameters describing electron spin relaxation, the
mean square of the zero field splitting, Δ2, the correlation time
for the modulation of the zero field splitting, τV298, while
The transverse 17O relaxation times were measured as a
function of temperature on aqueous solutions of the three
complexes. The longitudinal relaxation times were also
measured but were not included in the fittings as the differ-
ences between the paramagnetic solution and the diamagnetic
reference were too small to yield reliable values. The chemical
shifts were also measured but the differences in the chemical
shifts were too small compared to the line broadening of the
peak to be included in the treatment of the data.
1
The 17O transverse relaxation rates and the H NMRD data
at 298, 310, and 323 K (Fig. 3 and S4, S5†) were analysed simul-
taneously with the SBM theory to yield the microscopic para-
meters of the complexes characterizing water exchange and
rotation (see ESI† for equations). As can be seen in the figures,
the 17O reduced transverse relaxation rates (1/T2r) first increase
and then slightly decrease with increasing temperature, indi-
cating that the complexes are mainly in the slow to intermedi-
ate exchange regime. In the slow kinetic region, 1/T2r is
directly determined by the exchange rate kex. At higher
its activation energy, EV, has been fixed to 20 kJ mol−1
.
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Table 3 Best fit parameters obtained from the simultaneous analysis of 17O NMR and 1H NMRD data
Mn2ENOTAa
Mn(H2O)6
b
MnNODAHep
MnNODAHA
MnNODABA
kex298 (106 s−1
ΔH≠ (kJ mol−1
)
2.7 (2)
23.4 (9)
−24 (2)
25 (1)
84 (1)
60 (5)
70 (1)
30 (1)
2.7 (2)
23.5 (9)
−24 (2)
24 (1)
80 (1)
69 (9)
70 (1)
30 (1)
1.3 (3)
10.9 (9)
−72 (4)
27 (1)
121 (6)
60c
5.5
20.5
−28
18
2.1
32.9
+5.7
)
ΔS≠ (J mol−1 K−1
)
ER (kJ mol−1
τRO298 (ps)
τV298 (ps)
)
85
7.7
4.7
32.7
3.3
5.6
33.3
Δ2 (1018 s−1
)
70c
A0/ħ (106 rad s−1
)
33 (1)
a From ref. 15. b From ref. 52. c These values have been fixed for the fitting (see the text).
298
The diffusion coefficient DGdH
and its activation energy accounted for by the larger size of our complexes. The slightly
ED were fixed to 23 × 10−10 m2 s−1 and 20 kJ mol−1, respect- higher value observed for [Mn(NODABA)] can be explained by
GdH
ively. The distances between the Mn2+ ion and the inner and the higher rotational correlation time of the complex.
the outer sphere water protons were fixed to rMnH = 2.75 Å and
aMnH = 3.2 Å, respectively. The parameters resulting from the one water molecule in the first coordination sphere of the
best fit are presented in Table 3. As [Mn(NODABA)] is clearly metal ion (GdDOTA: r1 = 4.2 mM−1 s−1; GdDTPA: r1
These relaxivities are similar to those of Gd3+ chelates with
=
in the slow exchange region, no information on the electronic 4.3 mM−1 −1).41 In general, considering chelates of similar
s
parameters of these complexes can be obtained from the 17O size, Gd3+ complexes have higher relaxivities because of the
T2 values. Therefore, for the analysis of this system, we higher electron spin of Gd3+ with respect to Mn2+. A general
decided to fix the electronic parameters to the values obtained feature of Mn2+ complexes with respect to the Gd3+ analogues
from the fittings of the two other complexes.
The exchange rate of the complexes is of the same order of attain high relaxivities, but only for slowly rotating systems.
magnitude as that of the aqua ion and slightly lower than the We have also measured the NMRD profile of [Mn-
is their faster water exchange which can be advantageous to
one of the dinuclear complex ENOTA. The negative values of (NODAHep)] in the presence of HSA (Fig. S6 in ESI†). Given
the activation entropy tend to point to an associative mechan- the hydrophobic nature of the alkyl chain, non-covalent
ism for the exchange rate of the water molecule, as demon- binding between the complex and the protein through hydro-
strated for the Mn2ENOTA. The six-coordinated monohydrated phobic interactions is expected as it was reported for various
complexes will undergo an associative exchange which pro- Gd3+-54 and some Mn2+-chelates.9,10 Indeed, the NMRD curve
ceeds via a seven-coordinate transition state. [Mn(NODAHep)] displays a “hump” between 10 and 80 MHz, typical of slowly
and [Mn(NODABA)] are certainly six-coordinated species with rotating complexes (at 20 MHz, the relaxivity is nearly doubled
one water molecule in the first coordination sphere of Mn2+. in the presence of HSA) confirming that the complex is bound
In the case of [Mn(NODAHA)], the carboxylate function on the to HSA through hydrophobic interaction. We should note that
alkyl chain could also participate in the coordination sphere of this interaction is certainly not specific to HSA, but would be
Mn2+. Given the species found from potentiometric measure- operative for many other proteins as well.
ments (presence of a protonated complex) and the results
pH stability
obtained from the activation entropy, we can conclude that the
carboxylate function on the alkyl chain of NODAHA does not
participate in the coordination sphere of the metal ion. The
slower water exchange observed for [Mn(NODABA)] can be
explained by the presence of the bulky benzyl ring, which does
not facilitate the approach of the second water molecule in the
water exchange mechanism. The scalar coupling constants
found are typical of a Mn2+ complex,16,22 confirming the
monohydrated character of the complex.
The stability of [Mn(NODAHep)] was also investigated as a
function of the pH. The increase of relaxivity observed upon
going from basic to acidic pH can be attributed to the decom-
plexation of Mn2+ ion at acidic pH, lower than 5. This is
indeed in accordance with the species distribution obtained
from the stability constant of [Mn(NODAHep)] determined by
potentiometry (Fig. 4). We can conclude that in the pH range
7.4–9.3, the complex is formed.
The 1H NMRD profiles are comparable to those of Mn(EDTA)
and other monohydrated Mn2+ complexes. No second dispersion
is observed at low fields, unlike what has been observed for the
dinuclear Mn2ENOTA complex. This absence of second dis-
persion moreover confirms the absence of free Mn2+. The relaxivi-
ties of [Mn(NODAHep)], [Mn(NODAHA)] and [Mn(NODABA)] at
Conclusions
In this paper we introduce three new chelators designed
for Mn2+ as potential MRI CAs. The protonation constants
and the thermodynamic stabilities of their Mn2+ and Zn2+
chelates have been determined by pH potentiometry.
The thermodynamic stability constants are lower in
20 MHz and 298 K are respectively 4.73, 4.58 and 5.47 mM−1 s−1
,
remarkably higher than that of the similarly monohydrated Mn
(EDTA) (3.0 at 24 MHz, 298 K).53 This difference can be
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reactions were monitored by thin layer chromatography (TLC)
on aluminum plates coated with silica gel 60 F254 (Macherey-
Nagel). Chromatography separations were performed on
silica gel Whatman 230–240 Mesh. The NMR spectra were
recorded on a Varian Unity Plus 300 spectrometer or on a
1
BrukerAvance III 400 spectrometer. The H NMR spectra were
1
assigned using the two-dimensional COSY technique. The H
chemical shifts are reported in ppm, relative to tetramethyl-
silane (TMS) or sodium 2,2-dimethylsilapentane-5-sulfonate
(DSS) and the following abbreviations are used: s = singlet; d =
duplet; t = triplet; m = multiplet. pH measurements were per-
formed on a pH meter Crison micro TT 2050 with an electrode
Mettler Toledo InLab 422. Mass spectra (ESI+) were performed
on a VG Autospec M spectrometer or on a Finnigan LXQ MS
Detector.
Synthetic procedures
NODA(tBu)Hep. To
a solution of NO2AtBu (0.520 g,
1.45 mmol) in acetonitrile (22 mL), K2CO3 (0.606 g,
4.38 mmol) and 1-bromoheptane (0.230 mL, 1.46 mmol) were
added. The reaction mixture was stirred for 96 h. The reaction
mixture was filtered and the solvent removed under reduced
pressure. The oil obtained was purified by column chromato-
graphy using dichloromethane–ethanol 7 : 3 and dichloro-
methane–ethanol–NH3 7 : 3 : 0.5. The NODA(tBu)Hep (0.660 g,
1.45 mmol) was obtained as an oil with 100% yield. δH
(300 MHz; CDCl3; TMS) 0.85 (3 H, t, J = 6.8 Hz, (CH2)6CH3),
1.24 (8 H, m, (CH2)6CH3), 1.43 (20 H, m, (CH2)6CH3 and
C(CH3)3), 2.48 (2 H, t, J = 7.8 Hz, (CH2)6CH3), 2.70–2.90 (12 H,
Fig. 4 Stability of [Mn(NODAHep)] as a function of the pH: (a) paramagnetic
relaxation enhancement measured at 1 mM, 20 MHz, and 298 K. (b) Species dis-
tribution obtained under the same conditions from the stability constants
(Tables 1 and 2).
t
comparison to those of the analogous NOTA chelates. The
parameters governing relaxivity have been obtained by 1H
NMRD and 17O NMR studies. They are compatible with small
molecular weight chelates displaying one inner sphere water
molecule that exchanges via an associative water exchange
mechanism. The Mn2+ chelates display relaxivities between 4.6
and 5.5 mM−1 s−1 (20 MHz and 298 K), values which are
similar to those of monohydrated Gd3+ chelates. The slightly
higher relaxivity value found for [Mn(NODABA)] is explained
by a longer rotational correlation time.
m, en) and 3.29 (4 H, s, CH2CO2 Bu). δC (100.61 MHz; CDCl3;
TMS) 14.02 ((CH2)6CH3), 22.56 ((CH2)6CH3), 27.42 ((CH2)6CH3),
28.16 (C(CH3)3), 29.18 ((CH2)6CH3), 31.79 ((CH2)6CH3), 55.20
t
(en), 58.61 ((CH2)6CH3), 59.73 (CH2CO2 Bu), 80.65 (C(CH3)3)
t
and 171.46 (CH2CO2 Bu). HRMS (ESI+) calculated for
C25H50N3O4 (M + H)+ 456.38013. Found 456.37890.
NODAHep. Trifluoroacetic acid (30 mL) was added to a solu-
tion of NODA(tBu)Hep (0.660 g, 1.45 mmol) in dichloro-
methane (30 mL). The mixture was left stirring at room
temperature overnight. The solvent was removed under
reduced pressure and the oil obtained was washed with
hexane (3 × 15 mL) and water (3 × 15 mL). The NODAHep
(0.775 g, 1.13 mmol) was obtained as an oil with 78% yield. δH
(300 MHz; D2O; DSS) 0.84 (3 H, t, J = 6.8 Hz, (CH2)6CH3),
1.20–1.40 (8 H, m, (CH2)6CH3), 1.72–1.82 (2 H, m, (CH2)6CH3),
3.28–3.65 (14 H, m, (CH2)6CH3 and en) and 3.86 (4 H, s,
CH2CO2H). δC (75.43 MHz; D2O; DSS) 13.46 ((CH2)6CH3), 22.00
((CH2)6CH3), 23.80 ((CH2)6CH3), 25.78 ((CH2)6CH3), 27.98
((CH2)6CH3), 30.92 ((CH2)6CH3), 49.57 (en), 50.68 (en), 50.92
(en), 57.06 (CH2CO2H), 58.29 ((CH2)6CH3) and 173.29
(CH2CO2H). HRMS (ESI+) calculated for C17H34N3O4 (M + H)+
344.25493. Found 344.25378.
The alkylic pendant chain of NODAHep endows its Mn2+
chelate with amphiphilic character, forming micelles in solu-
tion. Its cmc has been determined by two distinct methods,
affording similar values (22.3 mM by fluorescence and
22.8 mM by relaxivity). Simultaneously the alkylic chain
permits the chelate to interact with HSA, resulting in a 60%
relaxivity increase (at concentrations similar to those present
in the blood, 4% (0.6 mM)55) due to an even longer tumbling
time. It is also expected that the interaction with HSA might
increase the retention time of the chelate in the blood stream.
Experimental
9-Fluorenylmethyl 4-(bromomethyl)benzoate
Materials and methods
N,N′-Dicyclohexylcarbodiimide (0.484 g, 2.34 mmol) and
Analytical grade reagents were purchased from Sigma-Aldrich, 4-(dimethylamino)pyridine (0.015 g, 0.123 mmol) were added
Fluka, Acros Organics, Macrocyclics and Chematech. The to
a
solution of 4-(bromomethyl)benzoic acid (0.505 g,
4528 | Dalton Trans., 2013, 42, 4522–4532
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2.35 mmol) and 9-fluorenemethanol (0.508 g, 2.59 mmol) in 6-bromohexanoic acid (2.002 g, 10.26 mmol) in 30 mL of
dry dichloromethane (15 mL) in an ice bath. The reaction methanol was added to the previous solution. The mixture was
mixture was stirred, at room temperature, for 72 h. The reac- left stirring at 313 K for 4 h. The solvent was evaporated giving
tion mixture was filtered and the solvent removed under methyl 6-bromohexanoate (1.903 g, 9.10 mmol) in 89% yield
reduced pressure. The residue obtained was purified by as a solid. δH (300 MHz; CDCl3; TMS) 1.46 (2 H, m, γCH2), 1.65
column chromatography eluted using cyclohexane–ethyl (2 H, m, βCH2), 1.86 (2 H, m, δCH2), 2.32 (2 H, t, J = 7.5 Hz,
acetate 4 : 1. The 9-fluorenylmethyl 4-(bromomethyl)benzoate αCH2), 3.39 (2 H, t, J = 6.6 Hz, εCH2) and 3.66 (3 H, s, CH3).
(0.436 g, 1.15 mmol) was obtained as an oil with 49% yield. δH
NODA(tBu)HA(me). K2CO3 (0.241 g, 1.741 mmol) and
(400 MHz; CDCl3; TMS) 4.40 (1 H, t, J = 7.2 Hz, Fm), 4.53 (2 H, methyl 6-bromohexanoate (0.148 g, 0.707 mmol) were added
s, C6H4CH2Br), 4.64 (2 H, d, J = 7.2 Hz, Fm), 7.34 (2 H, t, J = 7.6 to a solution of NO2AtBu (0.211 g, 0.589 mmol) in acetonitrile
Hz, Fm), 7.44 (2 H, t, J = 7.4 Hz, Fm), 7.52 (2 H, d, J = 7.8 Hz, (15 mL). The reaction mixture was stirred for 96 h. The reac-
C6H4CH2Br), 7.66 (2 H, t, J = 7.6 Hz, Fm), 7.81 (2 H, t, J = 7.6 tion mixture was filtered and the solvent removed under
Hz, Fm) and 8.07 (2 H, d, J = 7.8 Hz, C6H4CO2 Fm). δC reduced pressure. The oil obtained was purified by column
(100.61 MHz; CDCl3; TMS) 32.15 (PhCH2Br), 46.91 (CH2CH chromatography eluted using dichloromethane–ethanol 7 : 3
Fm), 67.12 (CH2CHFm), 120.08 (Fm), 125.02 (Fm), 127.17 (Fm), and dichloromethane–ethanol–NH3 7 : 3 : 0.5. The NODA(tBu)-
127.85 (Fm), 129.18 (C6H4CH2Br), 130.01 (C6H4CH2Br), 130.11 HA(me) (0.282 g, 0.581 mmol) was obtained with 99% yield, as
(C6H4CH2Br), 141.34 (Fm), 142.82 (C6H4CH2Br), 143.73 (Fm) an oil. δH (300 MHz; CDCl3; TMS) 1.31 (2 H, m, γCH2), 1.36 (18
and 165.90 (PhCO2Fm).
H, s, C(CH3)3), 1.57 (4 H, m, βCH2 and δCH2), 2.25 (2 H, t, J =
NODA(tBu)BA. To
a
solution of NO2AtBu (0.301 g, 7.2 Hz, αCH2), 2.62–3.10 (14 H, m, εCH2 and en), 3.28 (4 H, s,
t
0.842 mmol) in acetonitrile (20 mL), K2CO3 (0.472 g, CH2CO2 Bu) and 3.60 (3 H, s, CH3). δC (75.43 MHz; CDCl3;
3.42 mmol) and 9-fluorenylmethyl 4-(bromomethyl)benzoate TMS) 24.56 (δCH2), 26.02 (βCH2), 26.66 (γCH2), 28.10
(0.318 g, 0.838 mmol) were added. The reaction mixture was (C(CH3)3), 33.80 (αCH2), 51.40 (CH3), 53.98 (en), 54.83 (en),
t
stirred for several days. The reaction mixture was filtered and 57.33 (εCH2), 59.27 (CH2CO2 Bu), 80.83 (C(CH3)3), 171.60
the solvent removed under reduced pressure. The oil obtained (CH2CO2 Bu) and 173.92 (CO2CH3). HRMS (ESI+) calculated for
t
was purified by column chromatography eluted using ethyl C25H48N3O6 (M + H)+ 486.35431. Found 486.35278.
acetate–ethanol 1 : 1, ethanol and ethanol–NH3 10 : 0.5. NODA-
NODAHA. A solution of NaOH 1 M (7.5 mL) was added to a
(tBu)BA (0.410 g, 0.834 mmol) was obtained as an oil in 99% solution of NODA(tBu)HA(me) (0.225 g, 0.483 mmol) in meth-
yield. δH (300 MHz; CDCl3; TMS) 1.42 (18 H, s, C(CH3)3), anol (2.5 mL). The reaction mixture was left at reflux overnight.
t
2.79–3.47 (16 H, m, en and CH2CO2 Bu), 4.23 (2 H, s, The mixture was neutralized with 2 M HCl and stirred with
CH2C6H4CO2H), 7.50 (2 H, d, J = 7.8 Hz, CH2C6H4CO2H) and DOWEX for 5 h. The compound was removed with 0.1 M HCl
8.02 (4 H, d, J = 8.1 Hz, CH2 C6H4CO2H). δC (75.43 MHz; and the solvent evaporated under reduced pressure to give
CDCl3; TMS) 28.10 (C(CH3)3), 51.68 (en), 52.43 (en), 55.00 (en), NODAHA (0.218 g, 0.464 mmol), as an oil, in a 96% yield. δH
t
59.47 (CH2C6H4CO2H and CH2CO2 Bu), 81.24 (C(CH3)3), (400 MHz; D2O; DSS) 1.53 (2 H, m, γCH2), 1.77 (2 H, m, βCH2),
129.74 (CH2C6H4CO2H), 129.93 (CH2C6H4CO2H), 135.52 1.93 (2 H, m, δCH2), 2.54 (2 H, t, J = 7.6 Hz, αCH2), 3.44–3.75
t
(CH2C6H4CO2H), 136.55 (CH2C6H4CO2H), 170.73 (CH2CO2 Bu) (14 H, m, εCH2 and en), 3.94 (4 H, s, CH2CO2H). δC
and 171.11 (CH2C6H4CO2H). HRMS (ESI+) calculated for (100.61 MHz; D2O; DSS) 23.47 (δCH2), 23.78 (βCH2), 25.29
C26H42N3O6 (M + H)+ 492.30736. Found 492.30612.
(γCH2), 33.71 (αCH2), 49.99 (en), 50.77 (en), 51.09 (en), 57.84
NODABA. Trifluoroacetic acid (18 mL) was added to a solu- (εCH2), 58.12 (CH2CO2H), 174.29 (CO2H) and 178.93
tion of NODA(tBu)BA (0.415 g, 0.844 mmol) in dichloro- (CH2CO2H). HRMS (ESI+) calculated for C16H30N3O6 (M + H)+
methane (18 mL). The mixture was left stirring overnight and 360.21346. Found 360.21279.
the solvent removed under reduced pressure. The oil obtained
Potentiometric studies
was washed with hexane (3 × 9 mL) and water (3 × 9 mL).
NODABA (0.470 g, 0.651 mmol) was obtained in 77% yield, as Carbonate-free 0.1 M KOH and 0.1 M HCl were prepared from
an oil. δH (400 MHz; D2O; DSS) 3.25–3.63 (12 H, m, en), 3.81 Fisher Chemicals concentrates. Potentiometric titrations were
(4 H, s, CH2CO2H), 4.57 (2 H, s, CH2C6H4CO2H), 7.67 (2 H, d, J performed in 0.1 M aqueous KCl under a nitrogen atmosphere
= 6.3 Hz, CH2C6H4CO2H) and 8.06 (2 H, d, J = 6.3 Hz, and the temperature was controlled to 0.1 °C with a circulat-
CH2C6H4CO2H). δC (100.6 MHz; D2O; DSS) 49.36 (en), 50.31 ing water bath. The p[H] (p[H] = −log[H+], concentration in
(en), 50.74 (en), 56.32 (CH2CO2H), 60.37 (CH2C6H4CO2H), molarity) was measured in each titration with a combined pH
130.50 (CH2C6H4CO2H), 131.28 (CH2C6H4CO2H), 131.44 glass electrode (Metrohm) filled with 3 M KCl and the titrant
(CH2C6H4CO2H),
134.13
(CH2C6H4CO2H),
169.53 addition was automated by use of a 702 SM titrino system
(CH2C6H4CO2H) and 172.56 (CH2CO2H). HRMS (ESI+) calcu- (Metrohm). The electrode was calibrated in hydrogen ion con-
lated for C18H26N3O6 (M + H)+ 380.18216. Found 380.18152.
centration by titration of HCl with KOH in 0.1 M electrolyte
solution.56 A plot of meter reading versus p[H] allows the deter-
mination of the electrode standard potential (E°) and the slope
Methyl 6-bromohexanoate
Thionyl chloride (3.74 mL, 51.3 mmol) was slowly added factor (f). Continuous potentiometric titrations with HCl and
to methanol (15 mL) in an ice bath.
A
solution of KOH 0.1 M were conducted on aqueous solutions containing
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5 mL of NODABA 1.55 mM, 5 mL of NODAHA 1.34 mM, and maintained by a gas flow. The temperature was determined by
5 mL of NODAHep 3.08 mM in KCl 0.1 M, with 2 minutes previous calibration with a Pt resistance temperature probe.
waiting time between successive points. The titrations of the The longitudinal relaxation rates (1/T1) were determined in
metal complexes were performed with the same ligand solu- water. The least-squares fit of the 1H NMRD data was per-
tions containing 1 equiv. of the metal cation, with 2 minutes formed by using MicroMath Scientist version 2.0 (Salt Lake
waiting time between 2 points. Experimental data were refined City, UT, USA). The concentrations and pH of solutions were as
using the computer program Hyperquad 2008.57 All equili- follows: [Mn(NODAHep)] 4.68 mM; [Mn(NODABA)] 4.40 mM
brium constants are concentration quotients rather than activi- and [Mn(NODAHA)] 5.70 mM in 0.5 M HEPES buffer pH 7.4.
1
ties and are defined as:
Kmlh
The H NMRD profiles were obtained at 298, 310 and 323 K.
HRMS (ESI+): [Mn(NODAHep)] – calculated for C17H32MnN3O4
(M + H)+ 397.1773. Found 397.1761; [Mn(NODABA)] – calcu-
lated for C18H24MnN3O6 (M + H)+ 433.1046. Found 433.1039;
[Mn(NODAHA)] – calculated for C16H28MnN3O6 (M + H)+
413.1359. Found 413.1350.
½MmLlHhꢂ
h
¼
ð7Þ
m
l
½Mꢂ ½Lꢂ ½Hꢂ
The ionic product of water at 298 K and 0.1 M ionic
strength is pKw = 13.77.48 Fixed values were used for pKw,
ligand acidity constants and total concentrations of metal,
ligand and acid. All values and errors (one standard deviation)
reported are at least the average of two consistent and indepen-
dent experiments.
17O NMR studies
The longitudinal and transverse 17O relaxation rates (1/T1,2
)
and the chemical shifts were measured in aqueous solutions
of the various complexes in the temperature range 280–350 K,
on a Bruker Avance 500 (11.7 T, 67.8 MHz) spectrometer. The
temperature was calculated according to previous calibration
with ethylene glycol and methanol.59 An acidified water solu-
tion (HClO4, pH 3.3) was used as an external reference. Trans-
verse relaxation times (T2) were obtained by the Carr–Purcell–
Meiboom–Gill spin-echo technique60 and longitudinal relax-
ation times were measured by the inversion recovery
sequence.61 The technique of the 17O NMR measurements on
Gd3+ complexes has been described elsewhere.62 The samples
were sealed in glass spheres fitted into 10 mm NMR tubes to
avoid susceptibility corrections of the chemical shifts.63 To
improve the sensitivity, 17O-enriched water (10% H217O, Cor-
tectNet) was added to the solutions to reach around 1% enrich-
ment. The complex solutions were prepared in 0.5 M HEPES
buffer at pH 7, and the concentrations were as follows: [Mn-
(NODAHep] = 4 mM; [Mn(NODAHA)] = 4.04 mM; [Mn-
(NODABA)] = 4.00 mM. The 17O NMR data have been treated
according to the Solomon–Bloembergen–Morgan theory of
paramagnetic relaxation64 (see ESI†). The least-squares fit of
the 17O NMR data was performed using Micromath Scientist
version 2.0 (Salt Lake City, UT, USA). The reported errors corres-
pond to two times the standard deviation.
1H NMR titrations
Stock solutions of NODAHep (11.24 mM), NODABA
(10.92 mM), and NODAHA (10.15 mM) were prepared by dis-
solving the free chelator in D2O. The solution was titrated by
addition of small aliquots of 0.1 M NaOD or DCl solutions.
The final pH was corrected for the deuterium isotope effect
using the equation pH = pD − 0.4.58 Plots of chemical shifts
versus solution pH originated the acid–base titration curves.
Critical micellar concentration determination
Determination by fluorescence. A 40.5 mM stock solution of
[Mn(NODAHep)] was prepared in 0.1 M HEPES buffer pH 7.4.
This concentration was close to the limit of solubility of the
chelate. The estimation of the critical micellar concentration
(cmc) was performed using ANS (8-anilino-1-naphthalene sul-
fonic acid) as a fluorescence probe.44 The solutions used in
this study were prepared by dilution of the stock solution and
each one contained 1 × 10−5 M ANS. The fluorescence was
measured at 480 nm upon excitation at 350 nm at room temp-
erature. The fluorescence measurements were recorded on a
Bio-Tek® Synergy™ HT spectrofluorimeter using the software
KC4™.
Relaxometric measurements
A 41.34 mM stock solution of [Mn(NODAHep)] was prepared;
by dilution it was possible to have a set of solutions in the con-
centration range 4.68 mM–41.34 mM in 0.5 M HEPES buffer
pH 7.4, whose relaxivity was measured at 40 MHz and 298 K.
Interaction with HSA
A solution of [Mn(NODAHep)] (4.68 mM) and HSA (0.607 mM)
was prepared in 0.5 M HEPES buffer pH 7.4. The 1H NMRD
profile was obtained at 298 K.
1H NMRD studies
Proton NMRD (nuclear magnetic relaxation dispersion) pro-
files were recorded on a Stelar SMARtracer Fast Field Cycling
pH stability range
NMR relaxometer (0.01–10 MHz) and a Bruker WP80 NMR Solutions of [Mn(NODAHep)] (1 mM) were prepared in the pH
electromagnet adapted to variable field measurements and range 1.72–9.25. The longitudinal relaxation rates were
controlled by a SMARtracer PC-NMR console. The temperature measured at 20 MHz and 298 K. The measurements were
was monitored by a VTC91 temperature control unit and carried out in a BrukerMinispec MQ 20.
4530 | Dalton Trans., 2013, 42, 4522–4532
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Abbreviations
Acknowledgements
AAZTA
6-Amino-6-methylperhydro-1,4-diazepine triacetic
acid
8-Anilino-1-naphthalene sulfonic acid
Benzyloxymethyl
Critical micellar concentration
Contrast agents
Correlation spectroscopy
Duplet
Dichloromethane
Diphenylcyclohexylphosphate
1,4,7,10-Tetraazacyclododecane-1,7-diacetic
acid
1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic
acid
1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetate
1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetamide
N,N′-Dipyridoxylethylenediamine-N,N′-diacetate-
5,5′-bis-(phosphate)
We thank the financial support from Fundação para a Ciência
e a Tecnologia (F.C.T.), Portugal and Fundo Social Europeu
(FSE) (PhD grant SFRH/BD/63639/2009 and Rede Nacional de
RMN (REDE/1517/RMN/2005) for the acquisition of the Bruker
Avance III 400 NMR spectrometer at the University of Minho),
as well as financial support from La Ligue Contre le Cancer,
France (E. T.). This work was carried out in the frame of the
COST Actions D38 “Metal Based Systems for Molecular
Imaging” and TD1004 “Theragnostics Imaging and Therapy”.
ANS
BOM
cmc
CAs
COSY
d
DCM
diph
DO2A
References
DO3A
DOTA
DOTAM
DPDP
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Sodium 2,2-dimethylsilapentane-5-sulfonate
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Ethylenediamine tetraacetic acid
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Positive electrospray ionization
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4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
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HRMS
HSA
m
MeCN
MeOH
MRI
NMR
NMRD
NO2AtBu
High-resolution mass spectrometry
Human serum albumin
Multiplet
Acetonitrile
Methanol
Magnetic resonance imaging
Nuclear magnetic resonance
Nuclear magnetic relaxation dispersion
1,4,7-Triazacyclononane-1,4-diacetic acid
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NODABA
1,4,7-Triazacyclononane-1,4-diacetate-7-benzoic
acid
NODAHA 1,4,7-Triazacyclononane-1,4-diacetate-7-hexanoic
acid
NODAHep 1,4,7-Triazacyclononane-1,4-diacetate-
7-heptanil
NOTA
1,4,7-Triazacyclononane-1,4,7-triacetate
1,4,7-Triazacyclononane-1,4-diacetic
acid-7-octanoic acid
NOTAC8
s
Singlet
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SBM
t
Solomon–Bloembergen–Morgan
Triplet
TFA
TLC
TMS
Trifluoroacetic acid
Thin layer chromatography
Tetramethylsilane
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4532 | Dalton Trans., 2013, 42, 4522–4532
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