Optimising the relaxivities of Mn2+ complexes by targeting human serum albumin (HSA)

Article abstract of DOI:10.1039/c7dt01508a

We report two novel macrocyclic ligands based on the 1,4-DO2AM platform (1,4-DO2AM = 2,2′-(1,4,7,10-tetraazacyclododecane-1,4-diyl)diacetamide) and containing two benzyl groups attached either to the nitrogen atoms of the macrocyclic unit (1,4-BzDO2AM) or to the amide pendant arms (1,4-DO2AMBz). The protonation constants of the ligands and the stability constants of their Mn²⁺ complexes were determined using pH potentiometry. The introduction of benzyl groups results in a slight decrease of the stability constants of the Mn²⁺ complexes and a slight increase of their acid-catalysed dissociation reactions. A detailed relaxometric characterisation of the complexes using nuclear magnetic dispersion relaxation (NMRD) and ¹⁷O NMR studies indicated that the increase in molecular weight associated with the presence of benzyl groups results in a remarkable increase of proton relaxivities r_1p, which take values of 3.8, 3.5 and 2.5 mM^-1 s^-1 for [Mn(1,4-BzDO2AM)]²⁺, [Mn(1,4-DO2AMBz)]²⁺ and [Mn(1,4-DO2AM)]²⁺ (at 25 °C and 20 MHz). The [Mn(1,4-BzDO2AM)]²⁺ and [Mn(1,4-DO2AMBz)]²⁺ complexes form relatively strong adducts with Human Serum Albumin (HSA) with association constants of (3.9 ± 0.6) × 10³ and (2.0 ± 0.3) × 10³ M^-1, respectively. The interaction with the protein slows down the rotational tumbling of the complex in solution, which results in adducts endowed with remarkably high proton relaxivities (r_1p^b = 18.5 ± 0.7 and 27.4 ± 1.4 mM^-1 s^-1 for [Mn(1,4-BzDO2AM)]²⁺ and [Mn(1,4-DO2AMBz)]²⁺, respectively).

Full text of DOI:10.1039/c7dt01508a

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
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Optimising the Relaxivities of Mn2+ Complexes by Targeting
Human Serum Albumin (HSA)
Attila Forgács,a Lorenzo Tei,a Zsolt Baranyai,b David Esteban-Gómez,c Carlos Platas-Iglesiasc* and
Mauro Bottaa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We report two novel macrocyclic ligands based on the 1,4-DO2AM platform (1,4-DO2AM
= 2,2'-(1,4,7,10-
www.rsc.org/
tetraazacyclododecane-1,4-diyl)diacetamide) and containing two benzyl groups attached either to the nitrogen atoms of
the macrocyclic unit (1,4-BzDO2AM) or to the amide pendant arms (1,4-DO2AMBz). The protonation constants of the
ligands and the stability constants of their Mn2+ complexes were determined using pH potentiometry. The introduction of
benzyl groups results in a slight decrease of the stability constants of the Mn2+ complexes and a slight increase of their
acid-catalised dissociation reactions. A detailed relaxometric characterisation of the complexes using nuclear magnetic
dispersion relaxation (NMRD) and 17O NMR studies indicated that the increase in molecular weight associated with the
presence of benzyl groups results in a remarkable increase of proton relaxivities r1p, which take values of 3.8, 3.5 and 2.5
mM-1 s-1 for [Mn(1,4-BzDO2AM)]2+ [Mn(1,4-DO2AMBz)]2+ and [Mn(1,4-DO2AM)]2+ (at 25 ºC and 20 MHz). The
,
[Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+ complexes form relatively strong adducts with Human Serum Albumin
(HSA) with association constants of 3.9 + 0.6 × 103 and 2.0 + 0.3 × 103 M-1, respectively. The interaction with the protein
slows down the rotational tumbling of the complex in solution, which results in adducts endowed with remarkably high
b
proton relaxivities (r1p = 18.5 + 0.7 and 27.4 + 1.4 mM-1 s-1 for [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+
,
respectively.
MRI contrast agents.7,8 We recently have initiated a research
program to develop Mn2+ complexes that could represent an
alternative to the classical Gd3+ MRI contrast agents.9-12 We have
shown that Mn2+ complexes containing one or two water molecules
coordinated to the metal ion present relaxation efficiencies
comparable to those of the commercially available Gd3+ agents.11
Furthermore, we and others developed a family of macrocyclic and
non-macrocyclic ligands containing lipophilic units that form
micelles in solution and rather stable adducts with HSA, which
results in relatively high relaxivities due to the elongation of the
tumbling time of the complex in solution.13-15 Besides a potential
lower toxicity of the Mn2+ complexes compared to the Gd3+
counterparts, redox responsive agents based on the Mn2+/Mn3+ pair
were also proposed.16
Introduction
Derivatives
of
the
tetraaza
macrocycle
1,4,7,10-
tetraazacyclododecane (cyclen) functionalized with different type
and number of pendant arms have been investigated in detail in the
last decades due to several successful applications of their metal
complexes.1 For example, lanthanide complexes of such ligands
were proved useful as magnetic resonance imaging (MRI) contrast
agents,2 radiopharmaceuticals for nuclear medicine techniques,3
Chemical Exchange Saturation Transfer (CEST) agents4 and
luminescent probes.5 The tetraacetic acid derivative (DOTA) is
probably the most utilised, since it forms metal complexes
characterised by high thermodynamic stability and marked kinetic
inertness towards dissociation, hence favouring their use for in vivo
applications.6
Macrocyclic ligands often form metal complexes with higher
thermodynamic stability and kinetic inertness than related non-
macrocyclic analogues. In the context of MRI agents, the stable
complexation of the target metal ion is of great importance to avoid
the release of the toxic free metal ion in vivo. For instance, the
stability constants of Mn2+ complexes of cyclen-based ligands
containing acetate pendant arms were studied in aqueous solution
by means of potentiometric and microcalorimetric techniques,
which afforded logK, ∆H° and T∆S° values for the complexation
reactions. The metal complexes present high stability constants due
to both favourable enthalpic and entropic contributions. With the
unique exception of DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid, Scheme 1), the entropic term is prevalent.
Complexes of Mn2+ containing coordinated water molecules are
attracting increasing attention due to their potential application as
a. Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte
Orientale “A. Avogadro”, Viale T. Michel 11, 15121 Alessandria, Italy. E-mail:
mauro.botta@uniupo.it
b. Department of Inorganic and Analytical Chemistry, University of Debrecen, H-
4010, Debrecen, Egyetem tér 1., Hungary.
c. Universidade da Coruña, Centro de Investigacións Cient
íficas Avanzadas (CICA)
and Departamento de Química Fundamental, Facultade de Ciencias, 15071, A
Coruña, Galicia, Spain. E-mail: carlos.platas.iglesias@udc.es
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of the ligands; additional relaxometric and kinetic data; optimized Cartesian
coordinates obtained with DFT calculations. See DOI: 10.1039/x0xx00000x
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Furthermore, it was also noticed that 1,4-H2DO2A (1,4,7,10-
tetraazacyclododecane-1,4-diacetic acid) forms more stable
a
complex than the 1,7-H2DO2A isomer.17,18 The stability constant of
the Mn2+ complex with the bis-amide derivative 1,4-DOTAM was
found to be 2.5 log K units lower than that of [Mn(1,4-DO2A)].
However, the bis-amide ligand was found to be remarkably inert
with respect to dissociation, with a dissociation half-life at pH 7.4
ca. 10 times higher than that of [Mn(1,4-DO2A)].19
Scheme 2. Synthesis of the ligands. i) CH3CN, K2CO3, ꢀ, 24 h; ii)
Diethyloxalate, EtOH, ꢀ, 24 h; iii) Benzyl bromide, CH3CN, K2CO3, ꢀ, 24 h; iv)
5 M NaOH, ꢀ, 24 h; v) Dimethyl 2-chloroacetamide, CH3CN, K2CO3, ꢀ, 24 h.
Ligand protonation constants and stability constants of the Mn2+
complexes. The protonation constants of 1,4-DO2AMBz and 1,4-
BzDO2AM ligands, defined by Eq. (1), were determined by pH-
potentiometry in 0.1 M KCl aqueous solution. The logKiH values are
compared with those of 1,4-DO2AM, 1,4-DO2A2-, 1,7-DO2A2- and
DOTA4- in Table 1.
Scheme 1. Structures of the ligands discussed in this work.
We envisaged expanding the family of macrocyclic Mn2+
complexes based on cyclen while introducing hydrophobic units to
target HSA. Towards this aim, we present here the cyclen-based
ligands 1,4-DO2AMBz and 1,4-BzDO2AM (Scheme 1), which contain
two acetamide pendants at positions 1 and 4 of the cyclen ring and
two benzyl groups either at positions 7 and 11 of the macrocycle
(1,4-BzDO2AM) or as N-substitution of the acetamide pendants
(1,4-DO2AMBz). The presence of two hydrophobic moieties favours
the formation of adducts with Human Serum Albumin (HSA)20
[HiL]
KiH
=
with i = 1, 2,….., 6 (1)
[Hi−1L][L]
The protonation scheme of DO2AM has been studied in detail
1
using both H-NMR spectroscopy and pH-potentiometry methods.19
Because of the similarities of 1,4-BzDO2AM, 1,4-DO2AMBz and 1,4-
DO2AM, we can assume that the first protonation occurs on the
nitrogen atoms of the macrocycle N1 and N4, which are
functionalised with the acetamide pendant arms (the protonation
involves partially both N-atoms). The second protonation takes
place on the unsubstituted (1,4-DO2AMBz) or benzyl-substituted
(1,4-BzDO2AM) nitrogen atom N7 (or N10) to afford a trans bis-
protonated species on N1/N7 or N4/N10. The protonation of trans
nitrogen atoms minimises the electrostatic repulsion by allowing a
larger charge separation of the protonated sites.24
through non-covalent interactions. We report
a
detailed
investigation of the thermodynamic stability and dissociation
kinetics of the Mn2+ complexes, as well as a 1H and 17O NMR
relaxometric study to determine the parameters that govern the
relaxivity of these complexes. Finally, the interaction of the
complexes with HSA was assessed using relaxometric techniques.
Results and discussion
Synthesis of the Ligands. The 1,4-DO2AMBz ligand was
Comparison of the protonation constants of 1,4-BzDO2AM and
H
synthesized in two steps involving the preparation of sidearm 3 by 1,4-DO2AMBz with those of 1,4-DO2AM indicates that the logK1
reaction of 2-bromoacetyl bromide and benzyl amine,21 followed by value of 1,4-BzDO2AM is slightly higher, whereas the logK1 and
H
the regioselective cis alkylation of cyclen,22 which provided the 1,4- logK2 values of 1,4-DO2AMBz are significantly lower than those of
H
DO2AMBz ligand in ca. 20% yield over the two steps after semi 1,4-DO2AM. The basicity of the ring N-donor atoms might be
preparative HPLC purification. The 1,4-BzDO2AM ligand was influenced by four factors: i) the electron withdrawing effect of the
synthesised starting from cyclen, which was converted to 1,4- amide groups that reduces the basicity of the nitrogen donor
dibenzyl-1,4,8,11-tetraazacyclotetradecane following the method of atoms; ii) the electron donor or acceptor behaviour of the
Handel.23 Thus, cyclen was protected using diethyl oxalate to give substituents on the amide group; iii) the electrostatic repulsion
intermediate 4 (Scheme 2), which was subsequently alkylated with between the protonated ring N-donor atoms resulting in a lower
benzyl bromide using K2CO3 as a base. Basic deprotection of basicity of the ring amines and iv) the formation of H-bonding
compound 5 and alkylation of the resulting intermediate with N,N- interaction between the protonated nitrogen and the amide O-
dimethyl 2-chloroacetamide gave the 1,4-BzDO2AM ligand in a donor atoms that could increase the amine basicity.25 Likely, the
decent 20% yield over the four steps.
contribution of all these processes is responsible of the lower logKiH
values of the nitrogen donor atoms of 1,4-DO2AMBz as compared
2 | Dalton Trans., 2016, 00, 1-14
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to 1,4-DO2AM. The total basicity (Σ
logKiH) of 1,4-BzDO2AM is than expected by taking into account the ΣlogKiH values presented
comparable to that of 1,4-DO2AM, whereas the
Σ
logKiH value of in Table 1. Because of the similar basicity of the N donor atoms in
1,4-DO2AMBz is significantly lower (Table 1).
1,4-BzDO2AM and 1,4-DO2AM ligands, it can be assumed that the
The stability constants of the Mn2+ complexes of 1,4-BzDO2AM presence of the two bulky benzyl substituents introduces some
and 1,4-DO2AMBz are defined as in Eq 2:
steric constraints for the coordination of the ligand to the relatively
small Mn2+ ion (97 pm for coordination number six, high-spin
configuration).27
[ML]
(2)
KML
=
[M][L]
It should be noted that MnL complexes formed with 1,4-
BzDO2AM, 1,4-DO2AMBz and 1,4-DO2AM ligands are characterised
by lower stability constants than Mn(1,4-DO2A), Mn(1,7-DO2A),
Mn(DO3A) and Mn(DOTA) complexes. The lower logKML values of
the Mn2+-complexes formed with 1,4-DO2A-bisamide ligands can be
explained by the lower affinity of Mn2+ ion to the non-charged
amide oxygens with respect to the charged carboxylate O- donors
and/or the significantly lower basicity (ΣlogKiH) of the ring N-atoms.
The complexes of 1,4-BzDO2AM and 1,4-DO2AMBz form hydroxo
species at pH values above ca. 8.0 and 7.0, respectively (Figure 1).
The complexes dissociate at relatively high pH, with 2.6% of the
total Mn2+ being in the form of the aqua ion at pH 6.0 for the
Mn2+:1,4-BzDO2AM system and 22% for the Mn2+:1,4-DO2AMBz
system ([L] = [Mn2+] = 10-3 M). Thus, the [Mn(1,4-DO2AMBz)]2+
complex is somewhat more stable about neutral pH than the
[Mn(1,4-BzDO2AM)]2+ analogue. This is also reflected in the pMn
values (Table 1), which provide an indication of the stability of the
complex at pH 7.4.
The stability constants of the 1,4-BzDO2AM and 1,4-DO2AMBz
complexes have been calculated from the titration curves obtained
at 1:1 metal to ligand concentration ratios. The best fits of the
experimental data (volume of KOH added vs. pH) were obtained by
using the model that involves the formation of ML species at
equilibrium. However, the titration data of the Mn2+ - 1,4-BzDO2AM
and Mn2+ - 1,4-DO2AMBz systems indicated the consumption of
base at pH>8.5 due to the formation of hydroxide species. This
process was characterised by the equilibrium constant KMLH-1
defined according to Eq. (3):
[ML]
[MLH−1][H+ ]
(3)
KMLH
=
−1
The stability constants of the Mn2+ complexes formed with
1,4-BzDO2AM and 1,4-DO2AMBz are about 1 and 2 orders of
magnitude lower than that of [Mn(1,4-DO2AM)]2+, respectively. The
lower logKML value determined for [Mn(1,4-DO2AMBz)]2+ complex is
not surprising, as the total basicity of 1,4-DO2AMBz is about 2 logK
units lower than that of 1,4-DO2AM. However, the stability
constant of the [Mn(1,4-BzDO2AM)]2+ complex is significantly lower
Table 1. Ligand protonation constants and stability and protonation constants of the corresponding Mn2+ complexes determined using
potentiometric titrations (25 ºC, 0.1 M KCl) a
.
c
d
1,4-BzDO2AM
1,4-DO2AMBz
1,4-DO2AMb
1,4-DO2A2- c
1,7-DO2A2-
DOTA4-
11.41
9.83
H
H
H
H
H
H
logK1
logK2
logK3
logK4
logK5
logK6
11.11(1)
9.62(3)
10.14
11.40
9.58
3.74
1.65
-
11.66
9.75
4.06
1.78
-
8.22(4)
6.90(5)
8.38
-
-
-
4.38
-
-
-
4.63
-
-
-
-
-
-
1.92
-
-
1.58
∑logKiH
19.33
16.52
18.52
26.37
27.25
33.75
logKMnL
logKMnLH
logKMnLH2
11.54(4)
10.72(3)
12.64
15.22
15.07
4.09
3.70
-
19.33
-
-
-
-
-
-
-
4.15
-
-
-
-
-
logKMnLH
-1
10.44(5)
6.00
9.44(7)
6.69
pMne
6.94
7.03
6.75
9.02
a
e
Standard deviations (3σ) are shown in parentheses. b Reference 19. c Reference 17. d Reference 26. Defined as –log[Mn]free with pH = 7.4, [Mn2+] = [L] =
10-5 M.
Table 2. Rate constants and half-lives at pH=7.4 for the dissociation reactions of [Mn(1,4-BzDO2AM)]2+, [Mn(1,4-DO2AMBz)]2+ and related
complexes (0.1 M KCl, 25°C).
e
1,4-BzDO2AM
1,4-DO2AMBz
1,4-DO2AMd
1,4-DO2A2- e
1,7-DO2A2-
DOTA4- f
1.8 × 10–7
0.04
1.6 × 103
k0 (s-1)
k1 (M-1s-1)
k2 (M-2s-1)b
a
a
a
a
a
36 ± 2
38 ± 2
8.7
−
−
99
1.5 × 106
84
2.5 × 106
−
−
−
−
c
logKMnLH
4.15
4.48
4.26
kd (s-1) pH=7.4
1.4×10-6
136
1.5×10-6
126
3.5×10-7
556
3.9 ×10-6
49.4
3.3 × 10-6
58.3
2.6 ×10-6
1070
t1/2 (h) pH=7.4
a
b
The spontaneous dissociation of the complex characterised by k0 was found to be negligible. k2 rate constants characterizing the second-order
e
dependence on H+ concentration. Protonation constant characterising the protonated intermediate formed prior complex dissociation. Reference 19.
c
d
Reference 18. fe Reference 26.
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A 10 and 20-fold excess of Zn2+-ion was used in order to apply a
pseudo-first order kinetic model. Then, the rates of the reaction can
be expressed with Eq. (5):
d[MnL]
t
(5)
−
= kd [MnL]
tot
dt
Where kd is a pseudo-first-order rate constant and [MnL]t and
[MnL]tot are the concentrations of the MnL species at time t and the
total concentration of the complex, respectively. The calculated
pseudo-first order rate constants for the transmetallation reaction
of [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+ with Zn2+ as a
function of [H+] are shown in Figure 2 (see also Figures S7-S9, ESI†).
Fig. 1. Species distribution diagrams calculated for the 1,4-BzDO2AM:Mn2+
Fig. 2. kd values obtained for the transmetallation reactions of [Mn(1,4-
BzDO2AM)]2+ with Zn2+ ([MnL]=0.5 mM, [Zn2+]=10 mM (blue circles) and 20
mM (red triangles), 0.1 M KCl, 25°C).
(top) and 1,4-DO2AMBz:Mn2+ (bottom) systems. [L] = [Mn2+] = 10-3 M.
Transmetallation kinetics. Before in vivo application, it is
important to evaluate the kinetic inertness of any metal-complex
and thus the possible release of free metal ion or ligand in the
body.28 The kinetic inertness of a metal complex is characterised by
the rates of its dissociation, which is typically measured in strong
acidic conditions ([H+]>1.0 M).29 However, since these conditions
differ considerably from the physiological ones, it is crucial also to
consider the transmetallation reactions occurring with endogenous
metal ions (Cu2+, Zn2+ and Ca2+) at physiological pH. In fact, a Mn2+
complex in body fluids may interact with these ions resulting in the
release of the Mn2+-ion. Therefore, in order to assess the kinetic
inertness, the rates of the transmetallation reactions of Mn2+
complexes with Cu2+ and Zn2+ ions are typically determined in the
pH range 2-7.18,19,26 For Mn2+ DOTA-like complexes the metal ion is
slowly released through the proton-assisted dissociation pathway,
whereas the dissociation rate is generally not altered by the
The rates of the transmetallation reactions are directly
proportional to the H+ concentration and independent of the
concentration of Zn2+. The increase in the kd values with increasing
[H+] can be interpreted in terms of the relatively slow proton
assisted dissociation of the complex, followed by a fast reaction
between the free ligand and the exchanging Zn2+ ions. The
dependence of kd with [H+] can be expressed as a first-order
function of [H+] by taking into account the proton-independent (Eq.
(6)) and proton assisted pathways (Eq. (7)), which are characterised
by rate constants k0 and k1, respectively:
k0
MnL
Mn2+ + L
(6)
(7)
k1
MnL + H+
Mn2+ + HL
By taking into account all possible pathways the pseudo-first-
order rate constant (kd) can be expressed by the following linear
relationship:
presence of Zn2+ and Cu2+ 18,19,26
For the direct comparison of the
.
kinetic properties of [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-
DO2AMBz)]2+ with those of [Mn(1,4-DO2AM)]2+, [Mn(1,4-DO2A)],
[Mn(1,7-DO2A)] and [Mn(DOTA)]-, the method and the conditions
used were the same as those previously reported.18,19,26 Thus, the
rates of the transmetallation reactions of [Mn(1,4-BzDO2AM]2+ and
[Mn(1,4-DO2AMBz)]2+ were determined by relaxometry at 20 MHz
and 25°C in the pH range 4.5 – 6.5 using Zn2+ as exchanging metal
ion (Eq. (4); L denotes either 1,4-BzDO2AM or 1,4-DO2AMBz).
kd = k0 + k1 [H+]
(8)
The fit of the experimental kd values provided small values of k0
with large standard deviations, which indicates that the
spontaneous dissociation pathway does not contribute significantly
to the dissociation of the complex under the applied conditions. The
k1 rate constants characterising the proton-assisted dissociation of
the [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+ complexes
(Table 2) are about 4 times higher than that of the [Mn(1,4-
MnL + Zn2+ ꢀ ZnL
+
Mn2+
(4)
4 | Dalton Trans., 2016, 00, 1-14
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DO2AM)]2+, but about three times smaller than those of [Mn(1,4- whole temperature range investigated is a clear indication of a fast
DO2A)] and [Mn(1,7-DO2A)] complexes. It is generally accepted rate of exchange for the coordinated water molecule, in agreement
that the proton assisted dissociation of Mn2+ complexes of DOTA- with the qualitative analysis of the NMRD profiles recorded at
like ligands containing carboxylate pendant arms takes place by the different temperatures.
protonation at one of the carboxylate groups, which is then likely
followed by proton transfer to the ring nitrogen, resulting in the
cascade-like de-coordination of each donor atom with the release
of Mn2+ from the coordination cavity.26 Since the replacement of
the protonable carboxylate groups with non-protonable amide
groups does not allow the formation of the protonated MnHL
intermediate, it is likely that the proton assisted dissociation of
Mn2+ complexes formed with DO2A-bisamides takes place by
protonation of the ring N-atom followed by the release of the Mn2+
ion. By taking into account the k1 values of [Mn(1,4-BzDO2AM)]2+,
[Mn(1,4-DO2AMBz)]2+ and [Mn(1,4-DO2AM)]2+, it appears that the
protonation of the ring N-atom in [Mn(1,4-BzDO2AM)]2+ and
[Mn(1,4-DO2AMBz)]2+ occurs more easily, which might be explained
by a weaker interaction between the N donor atoms of 1,4-
BzDO2AM and 1,4-DO2AMBz ligands and Mn2+. This hypothesis is in
line with the lower stability constants of [Mn(1,4-BzDO2AM)]2+ and
[Mn(1,4-DO2AMBz)]2+ complexes (Table 1). The half-life (t1/2) of
dissociation of [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+
calculated for pH=7.4 is about four times lower than that measured
for [Mn(1,4-DO2AM)]2+, but remains significantly longer than those
reported for [Mn(1,4-DO2A)] or [Mn(1,7-DO2A)].
1H and 17O NMR relaxometric studies. The r1p values of [Mn(1,4-
BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+ as measured at 25°C, 20
MHz and neutral pH are 3.8 and 3.5 mM-1 s-1, respectively. These
values are considerably higher than that of [Mn(1,4-DO2A)] and
[Mn(1,4-DO2AM)]2+,9,19 suggesting that both complexes feature one
coordinated water molecule and an enhanced inner sphere
relaxivity associated with their larger size (longer τR). NMRD profiles
were measured at 298 and 310 K over the proton Larmor frequency
range 0.01-70 MHz, corresponding to magnetic field strengths
varying between 2.343×10-4 T and 1.645 T (Fig. 3). The profiles have
a rather simple functional form typical for low molecular weight
complexes, displaying a region of constant relaxivity at low fields
and a simple dispersion around 6-8 MHz, with a second region at
high fields (> 20 MHz) where the relaxivity tends to flatten out. The
absence of a second dispersion at low fields (~1 MHz) indicates that
the scalar contribution to relaxivity is negligible.30
Fig. 3. 1H NMRD profiles recorded at different temperatures for [Mn(1,4-
BzDO2AM)]2+ (top) and [Mn(1,4-DO2AMBz)]2+ (bottom). The lines represent
the fit of the data as explained in the text.
The 1H NMRD and 17O NMR data were fitted simultaneously
according to the established theory of paramagnetic relaxation
expressed by the Solomon-Bloembergen-Morgan32 and Freed’s33
equations for the inner- (IS) and outer sphere (OS) proton relaxation
mechanisms, respectively, and of the Swift-Connick theory for 17O
As for the parent [Mn(1,4-DO2AM)]2+ complex, relaxivity
decreases by increasing temperature over the entire frequency
range, clearly indicating that both Mn2+ chelates are in the fast-
exchange regime and that r1p is only limited by the rotational
correlation time. Given the relatively large number of parameters
influencing the relaxivity of Mn2+ complexes, the water exchange
kinetics of the coordinated water molecules were investigated by
analysing the temperature dependence of the 17O NMR transverse
relaxation rates (R2) and paramagnetic chemical shifts (Δω). The
experimental data were measured on a high-resolution NMR
spectrometer, operating at 11.7 T, on aqueous solutions of the
complexes (2-7 mM) at neutral pH. The data were then analysed
according to the well-established set of Swift-Connick equations.31
The reduced R2 (1/T2r) and Δω values (Δωr) are presented in Figure
4. The increase of 1/T2r with decreasing temperature over the
relaxation.31 Following
a
well-established practice, some
parameters were fixed to reasonable values due to the large
number of parameters that must to be considered during the fit:
the hydration number q was fixed to 1; the distance between the
metal ion and the protons of the bound water molecule, r, was
fixed to 2.83 Å; a and D were set to 3.6 Å and 2.3×10-5 cm2 s-1 (at
25°C), respectively; EV was fixed to 1.0 kJ mol-1. The values of ER
(activation energy for the rotational motion of the complex) and ED
(activation energy of D) were fixed to those found for [Mn(1,4-
DO2A)]: 19.1 and 17.3 kJ mol-1, respectively. The relevant best-fit
parameters are listed in Table 4 and compared with those of related
macrocyclic Mn2+ complexes.
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Table 3. Parameters obtained from the simultaneous analysis of 17O NMR and 1H NMRD data
.
1,4-BzDO2AM
1,4-DO2AMBz
1,4-DO2AMb
2.5/2.0
111
1,4-DO2Ac
2.1/1.7
r1p at 25/37 ºC / mM-1 s-1 b
3.8/2.8
3.5/2.8
298 / 106 s-1
kex
253 + 9
175 + 5
1134
29.4
46
ꢀH‡ / kJ mol-1
39.8
14.4 + 1.1
96 + 2
24.6 + 0.8
85 + 3
τR298
/ ps
53
298 / ps
5.5
1.0a
4.4
1.0a
τv
13 + 1
1.0a
11 + 1
1.0a
Ev / kJ mol-1
ꢀ2 / 1019 s-2
AO/ħ/ 106 rad s-1
rMnH/Å
51
48
20 + 2
-31.0 + 0.4
2.83a
3.6a
20 + 1
-39.0
2.83a
3.6a
0.87a
-43
-33.0 + 0.3
2.83a
3.6a
0.87a
2.83a
aMnH/Å
q298
3.6a
1a
1a
a Parameters fixed during the fitting procedure. bData from Ref 19. cData from Ref 9.
six- and seven-coordinate states. The seven-coordinate complexes
investigated here are expected to follow a dissociatively activated
water exchange reaction proceeding through a six-coordinated
transition state. Most likely, the presence of the benzyl groups
introduces some steric hindrance around the coordinated water
molecule, which facilitates its departure in a dissociatively activated
mechanism.
DFT calculations. DFT calculations were used to gain insight into
the structure of the Mn2+ complexes of 1,4-BzDO2AM and 1,4-
DO2AMBz in solution. Geometry optimizations were performed on
the
[Mn(1,4-BzDO2AM)(H2O)]2+·2H2O
and
[Mn(1,4-
DO2AMBz)(H2O)]2+·2H2O systems, which contain two second-sphere
water molecules besides the water molecule directly coordinated to
the metal ion. The explicit inclusion of two second-sphere water
molecules was found to be critical for an accurate calculation of
Mn-Owater distances and 17O hyperfine coupling constants AO/ħ.11,34
Our calculations provided optimized geometries (Figure 5) with
coordination environments very similar to those calculated
previously for [Mn(1,4-DO2A)(H2O)],9 and that observed in the solid
state for [Mn(1,4-DO2A)].17 Benzylation of the nitrogen atoms of
the macrocycle provokes a significant lengthening of the Mn-N
distances, which are significantly longer in the complex of 1,4-
BzDO2AM compared to the 1,4-DO2AMBz derivative. The spin
densities calculated at the nuclei of the coordinated water
molecules provide AO/ħ values of -52.5 × 106 and -47.3 × 106 rad s-1
for the complexes of 1,4-BzDO2AM and 1,4-DO2AMBz, respectively.
The calculated values are somewhat higher than those determined
experimentally from the analysis of the 17O NMR data, but still
similar to those reported for different Mn2+ complexes. This could
be related to the difficulties in the experimental determination of
AO/ħ values from the observed 17O NMR shifts, or perhaps to the
fact that our DFT calculations neglected dynamic effects.
Alternatively, a certain fraction of q = 0 complexes in solution could
explain the lower experimental AO/ħ data.
Figure 4. Reduced transverse 17O NMR relaxation rates and 17O NMR
chemical shifts measured for[Mn(1,4-DO2AMBz)]2+ (circles) and [Mn(1,4-
BzDO2AM)]2+ (squares) at 11.74 T. The lines represent the fit of the data as
explained in the text.
The electron relaxation parameters, ꢀ2 and
both macrocyclic complexes and similar to the values found for the
parent complex. This represents strong indication of the
τV, are very similar for
a
occurrence of strictly analogous solution structures for the three
complexes. The rotational correlation time τR assumes values of 96
and 85 ps for [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+
,
respectively. This nicely reflects the increased molecular mass of
the benzyl substituted derivatives over the parent complex and
confirms the limiting role of the rotational dynamics to the
relaxivity of the complexes. In addition, the difference in the r1p and
τR values between the two chelates is well accounted for by their
slightly different molecular masses.
The rates of water exchange are about twice that of [Mn(1,4-
DO2AM)]2+ and approximately half that of [Mn(EDTA)]2-. Thus, the
introduction of hydrophobic substituents affects not only the
rotational dynamics but also the water exchange dynamics of the
complexes, likely by influencing the relative energy values of their
6 | Dalton Trans., 2016, 00, 1-14
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hypothesise that the presence of the two hydrophobic groups on
the coordinating acetamide moieties limits the degree of local
motions involving the coordination cage (longer effective rotational
correlation time) and thus increases relaxivity. The association
constants obtained for the Mn2+ complexes reported here are 2-3
times higher than those reported for Gd3+ complexes bearing
analogous targeting groups.35 However, these anionic Gd-chelates
differ from the (cationic) Mn2+ complexes reported here in their
overall charges.
A small but detectable relaxivity enhancement in the presence of
HSA is observed also for 1,4-MnDO2AM (Figure 6). In this case, only
electrostatic forces govern the weak interaction and the binding
sites are localized on the negatively charged domain of the protein.
The limited relaxivity gain (Table 4) suggests either a marked
decrease of the rate of water exchange or a restricted access of
bulk solvent molecules to the paramagnetic site. Clearly, the
binding sites and the mechanisms of the relaxation enhancement
are quite different for the parent 1,4-MnDO2AM complex and the
bis-benzyl derivatives. For these latter, the hydrophobic interaction
predominates and drives the protein binding event.
Table 4. Best-fit parameters obtained from the analysis of the 1H
relaxometric titrations (20 MHz; 298 K) of the Mn2+ complexes 1,4-
BzDO2AM, 1,4-DO2AMBz and 1,4-DO2AM with HSA.
1,4-BzDO2AM 1,4-DO2AMBz 1,4-DO2AM
n·KA (M-1)
r1pb (mM-1 s-1)a
r1pf (mM-1 s-1)b
3909 ± 583
18.5 ± 0.7
3.8
1964 ± 342
27.4 ± 1.4
3.5
1213 ± 95
5.6 ± 0.5
2.5
a Relaxivity of the complex fully bound to the protein. b Relaxivity of the free
(unbound) complex.
Figure 5. Structures of the [Mn(1,4-BzDO2AM)(H2O)]2+·2H2O (top) and
[Mn(1,4-DO2AMBz)(H2O)]2+·2H2O (bottom) systems obtained with DFT
calculations (M062X/TZVP). Bond distances of the metal coordination
environments are given in Å.
Human Serum Albumin (HSA) binding studies. The presence of
hydrophobic groups in the structure of the 1,4-BzDO2AM and
1,4-DO2AMBz allows the complexes to bind HSA. The binding
interaction has been investigated using the well-established proton
relaxation enhancement (PRE) technique.14 This method consists in
measuring the increase of the water proton longitudinal relaxation
rate (R1) as a function of increasing concentration of the protein (at
20 MHz and 298 K in the present case, Figure 6). R1 is enhanced by
the increase of the fraction of bound complex, characterized by a
longer rotational correlation time. The fitting of the experimental
data provides the values of the thermodynamic association
constant, KA, the number of the equivalent and independent
binding sites, n, and the relaxivity of the resulting paramagnetic
adduct, r1bound. All the data were fitted to 1:1 binding isotherms
even though the presence of multiple affinity sites on HSA cannot
be excluded for these complexes. The affinity of the complexes for
the protein is influenced by the position of the pendant
hydrophobic moieties, with the association constant KA obtained for
[Mn(1,4-BzDO2AM)]2+ being ca. twice that obtained for [Mn(1,4-
DO2AMBz)]2+ (Table 4). However, the relaxivity of the fully bound
form is considerably higher for the adduct formed by [Mn(1,4-
DO2AMBz)]2+, which indicates that the relative position of the
benzylic groups in the two complexes influences the motional
coupling between the paramagnetic unit and the protein. We
Figure 6. Changes in the observed longitudinal relaxation rates of water
protons observed upon addition of HSA to solutions of the
[Mn(BzDO2AM)]2+ (0.139 mM), [Mn(DO2AMBz)]2+ (0.199 mM) and [Mn(1,4-
DO2AM)]2+ (0.298 mM) complexes. The solid lines represent the least-
squares fits of the data according to a 1:1 binding isotherm.
Conclusions
The chemical modification of the basic structure of DO2AM, with
the introduction of two benzyl groups either on the macrocycle or
on the pendant arms, has a strong effect on various properties of
the corresponding Mn2+ complexes. First, the complexes of the
benzyl derivatives show higher r1p values. The increase of molecular
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mass appears to be translated entirely into an increase of relaxivity, 11.5 mmol) and benzyl amine (1.14 mL, 10.4 mmol) were dissolved
thus suggesting a rather compact structure characterized by an in CH2Cl2 (10 mL) and triethylamine (0.88 mL, 6.26 mmol) was
o
isotropic tumbling motion. The high relaxivity values suggest the added at 0 °C. The reaction mixture was stirred at 0 C for 1 h, and
presence of one bound water molecule, although we cannot then allowed to warm to room temperature. Water (15 mL) was
exclude the presence of a small population of the q=0 isomer. In added and the aqueous layer was extracted with CH2Cl2 (4x 15 mL).
any case, the hydration state of the parent complex did not The combined organic layers were washed with 5% HCl (50 mL),
decrease upon the chemical modification. Second, the rate of water water, a saturated NaHCO3 solution (50 mL) and brine. The organic
exchange has increased by a factor of ca. two as compared to the layer was dried over MgSO4 and then filtered. The solvent was
value of [Mn(DO2AM)]2+. This is likely related to the steric removed in vacuum and the crude product 3 was purified by flash
compression introduced by the benzyl groups around the water chromatography (petroleum ether:Ethyl acetate 7:3) on silica gel.
binding site. Third, the presence of the hydrophobic pendant The formation of N-benzyl-2-bromoacetamide (3)21 was confirmed
groups enables the formation of non-covalent adducts with HSA, by mass spectrometry. ESI+/MS: m/z 228.0 ([C9H11BrNO]+). Cyclen
b
which are characterised by affinity constants and r1p quite (0.28 g, 1.6 mmol) was dissolved in acetonitrile (15 mL) and K2CO3
comparable to those typical of the analogous Gd3+ complexes.
(0.33 g, 2.4 mmol) was added. A solution of compound 3 (0.74 g 3.3
The results reported in this work also highlight the difficulties to mmol) in the same solvent (20 mL) was added dropwise. The
design Mn2+ complexes containing a coordinated water molecule mixture was heated to reflux for 24 h. Then, the solution was
and possessing
a high thermodynamic stability and kinetic filtered to remove the excess of carbonate, and the solvent was
inertness, which represents a challenge for coordination chemistry. removed. The resulting aqueous solution was dried and the crude
The introduction of benzyl groups was found to be detrimental for product was then purified by semi-preparative HPLC-MS (Solvent A:
both the thermodynamic stability and kinetic inertness. These H2O TFA 0.1%; Solvent B: MeOH; 0-2 min 10% B then gradient 10-
Mn(II) chelates are cationic and thus not suitable for in vivo 100% B in 18 min; Flow 20 mL/min; retention time: 6.3 min) to
applications. Previous data on lanthanide complexes of DOTA- obtain 140 mg of an orange oil (0.26 mmol, 18 % yield). ESI-MS
tetraamide derivatives indicated that at low doses the compounds (m/z): found 467.5 [M + H+] (calc. for C26H38N6O2: 466.5). 1H-NMR
are not easy to dissociate in vivo and are well tolerated.36 This is (D2O, 500 MHz): δ 3.22 (σ CH2NH 4H) 3.05 (s, CH2CO, 4H), 2.85-2.65
due to their high degree of kinetic inertness. In order for the Mn(II) (m, NCH2 ring, 16H), 7.51-7.41 (m, CHAr, 10H); 13C-NMR (D2O, 125
complexes can be employed at high doses in vivo, the kinetic inertia MHz): δ 171.4 (C=O), 131.0 (CH), 129.1 (CH), 57.7 (CH2), 53.5 (CH2),
and the incorporation of negatively charged groups are important 53.0 (CH2), 50.0 (CH2), 48.9 (CH2), 36.6 (CH2).
requirements.
Synthesis of 2,2'-(7,10-dibenzyl-1,4,7,10-tetraazacyclododecane-
We are currently exploring chemical modifications of these and 1,4-diyl) bis(N,N-dimethylacetamide) (1,4-BzDO2AM). Cyclen (0.5 g
related ligands to obtain efficient and safer Mn2+-based MRI probes. 2.9 mmol) was dissolved in ethanol (15 mL) and diethyl oxalate (585
μL, 4.3 mmol) was added dropwise. The mixture was heated to
reflux for 24 h and then the solvent was removed in vacuum to give
Experimental Section
intermediate
1,4,7,10-tetraazabicyclo[8.2.2]tetradecane-11,12-
dione (4): ESI+/MS: m/z 227.2 ([C10H19N4O2]+). Compound 4 (0.64 g,
2.8 mmol) was dissolved in acetonitrile (15 mL) and benzyl bromide
(840 μL 7.0 mmol) and K2CO3 (2.35 g 17 mmol) were added to the
mixture. The mixture was heated to reflux for 24 h and then the
solvent was removed in vacuum. The crude product 4,7-dibenzyl-
1,4,7,10-tetraazabicyclo[8.2.2]tetradecane-11,12-dione (5) was
dissolved in CH2Cl2 (20 mL) and washed with water (3x15 mL). The
product was purified by flash chromatography (98:2 DCM:MeOH,
silica gel). ESI-MS (m/z): found 407.5 [M + H+]. Compound 5 was
dissolved in 5 M NaOH (20 mL) and the mixture stirred at 100 °C for
24 h. The solution was concentrated in a rotary evaporator to a
volume of 10 mL and washed with CH2Cl2 (3x20 mL). The combined
organic extracts were washed with water (3x20 mL). The organic
layer was dried over MgSO4 and then filtered and concentrated
General. All chemicals were purchased from Sigma-Aldrich Co. and
were used without further purification. The concentration of the
MnCl2 and ZnCl2 solutions were determined by complexometric
titration with standardized Na2H2EDTA and xylenol orange (ZnCl2) or
Eriochrome Black T (MnCl2) as indicators. The concentration of the
solutions of 1,4-BzDO2AM and 1,4-DO2AMBz was determined by
pH-potentiometric titration in the presence and absence of a 50-
fold excess of CaCl2. The pH-potentiometric titrations were carried
out with a standardised 0.2 M KOH solution. 1H and 13C NMR
spectra were recorded on a Bruker DRX 400 (9.39 T) and Bruker
Avance III (11.74 T) spectrometers. Chemical shifts are reported
relative to TMS and were referenced using the residual proton
solvent resonances.37 Electrospray ionization mass spectra (ESI MS)
were recorded on an SQD 3100 Mass Detector (Waters), operating
in positive or negative ion mode, with 1% v/v HCOOH in methanol
as the carrier solvent. HPLC analyses were carried out on a 1525EF
Waters liquid chromatograph equipped with Waters 2489 UV/vis
and Waters SQD 3100 MS detectors and using a Waters Atlantis® T3
RPC18 column (150 mm × 4.6 mm, 5 μm). Preparative HPLC
separations were carried out using a Waters Atlantis® T3 OBD RPC18
column (100 mm × 19 mm, 5 μm) and a Waters FCIII fraction
collector.
under vacuum. ESI-MS (m/z): found 353.5 [M
+
H+]. The
intermediate was used without any further characterization.23
Compound 6 (0.17 g 0.5 mmol) and K2CO3 (0.63 g, 4.5 mmol) were
suspended in ACN (10 ml) and N,N-dimethyl-2-chloroacetamide
(105 μL 1.01 mmol) was added dropwise. The mixture was stirred
for 24 h and then the solvent was removed in vacuo. The crude
product 8 was purified by flash chromatography (DCM:MeOH 99:1,
silica gel) to obtain a pale yellow oil. (0.46 mmol, 16 % yield). ESI-
MS (m/z): found 523.5 [M + H+] (calc. for C30H46N6O2: 522.5). 1H-
NMR (D2O, 500 MHz): δ 4.31 (σ CH2 4H) 3.18 (s, CH2CO, 4H), 3.51 (s
Synthesis of 2,2'-(1,4,7,10-tetraazacyclododecane-1,4-diyl)bis(N-
benzylacetamide) (1,4-DO2AMBz). 2-Bromoacetyl bromide (1 mL,
8 | Dalton Trans., 2016, 00, 1-14
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calculations were performed using the computer program
Micromath Scientist, version 2.0 (Salt Lake City, UT, USA).
CH3 12H) 2.01-2.28 (m, NCH2 ring, 16H), 7.30-7.37 (m, CHAr, 10H);
13C-NMR (D2O, 125 MHz): δ 172.5 (C=O), 137.8 (CH), 126.5.0 (CH),
127.0 (CH), 127.6 (CH) 49.5 (CH2), 50.1 (CH2), 50.6 (CH2), 51.2 (CH2),
51.9 (CH2), 55.0 (CH2) 41.9 (CH3).
1H NMRD and 17O NMR Measurements. The water proton
longitudinal relaxation rates as a function of pH (20 MHz) were
measured with a Stelar Spinmaster Spectrometer FFC−2000 (Mede,
PV, Italy) on about 0.6-2.0 mM aqueous solutions in non-deuterated
water. The exact concentrations of Mn2+ ions were determined by
measurement of bulk magnetic susceptibility shifts of a tBuOH
signal on a Bruker Avance III spectrometer (11.7 T).40 The 1H T1
relaxation times were acquired by the standard inversion recovery
method with a typical 90° pulse width of 3.5 μs, 16 experiments of 4
scans each. The reproducibility of the T1 data was ±5%. The
temperature was controlled with a Stelar VTC-91 airflow heater
Equilibrium Measurements. The protonation constants of 1,4-
BzDO2AM and 1,4-DO2AMBz, as well as the stability and
protonation constants of the Mn2+ complexes formed with the 1,4-
BzDO2AM and 1,4-DO2AMBz were determined by pH-
potentiometry. The pH-potentiometic titrations were performed at
1:1 metal-to-ligand concentration ratios (the concentration of the
ligand was generally 0.002 M). For the calculation of the equilibrium
constants the mL base – pH data were used, obtained in the pH
range 1.7–12.0. For the pH measurements and titrations,
a
Methrohm 888 Titrando titration workstation and a Metrohm
6.0233.100 combined electrode were used. Equilibrium
measurements were carried out at a constant ionic strength (0.1 M
KCl) and 25 °C in 8 mL samples. The solutions were stirred, and N2
was bubbled through them. KH-phthalate (pH=4.005) and borax
(pH=9.177) buffers were used to calibrate the pH meter. For the
calculation of [H+] from the measured pH values, the method
proposed by Irving et al. was used.38 A 0.01M HCl solution was
equipped with
a calibrated copper–constantan thermocouple
(uncertainty of ±0.1 °C). The proton 1/T1 NMRD profiles were
measured on a fast field-cycling Stelar SmartTracer relaxometer
over a continuum of magnetic field strengths from 0.00024–0.25 T
(corresponding to 0.01–10 MHz proton Larmor frequencies). The
relaxometer operates under computer control with an absolute
uncertainty in 1/T1 of ±1%. Additional data points in the range 15–
titrated with the standardized KOH solution in the presence of 0.1 70 MHz were obtained on a Stelar Relaxometer equipped with a
M KCl ionic strength, respectively. The differences between the
measured (pHread) and calculated pH (-log[H+]) values were used to
obtain the equilibrium H+ concentration from the pH values,
measured in the titration experiments. The ion product of water
was determined from the same titrations (HCl/KOH) using the data
in the pH range of 11.5-12.0. Equilibrium constants were calculated
with the PSEQUAD program.39
Bruker WP80 NMR electromagnet adapted to variable-field
measurements (see above).
Variable-temperature 17O NMR measurements were recorded
on a Bruker Avance III spectrometer (11.7 T) equipped with a 5 mm
probe and standard temperature control unit. Aqueous solutions of
the complexes (4 mM) containing 2.0% of the 17O isotope
(Cambridge Isotope) were used. The observed transverse relaxation
rates were calculated from the signal width at half-height.
DFT calculations. Full geometry optimizations of the
Kinetic studies. The kinetic inertness of the Mn2+ complexes was
characterised by the rates of the exchange reactions taking place
between the MnL complexes and Zn2+. The rates of the metal
exchange reactions of [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-
DO2AMBz)]2+ with Zn2+ was followed by measuring the water
[Mn(1,4-DO2AMBz)(H2O)]2+·2H2O
and
[Mn(1,4-BzDO2AM)(H2O)]2+·2H2O systems were performed
employing DFT calculations at the M062X/TZVP41,42 level with the
Gaussian 09 package (Revision D.01).43 Solvent effects were
included by using the polarizable continuum model (PCM), in
particular the integral equation formalism (IEFPCM) variant.44 No
symmetry constraints have been imposed during the optimizations.
The stationary points found on the potential energy surfaces as a
result of geometry optimizations were characterized via frequency
analysis. Hyperfine coupling constants were calculated with the
same computational approach using the EPR-III basis set of Barone
for C, H, O and N.45
proton relaxation rates (1/T1) of the samples with
a Stelar
Relaxometer equipped with a Bruker WP80 NMR electromagnet
adapted to variable-field measurements (15-80 MHz proton Larmor
frequencies). The kinetic measurements were performed on the
Stelar Relaxometer at 0.47 T (20 MHz) and 25 °C owing to the large
differences in the relaxivities of [Mn(1,4-BzDO2AM)]2+ (r1p=3.8
mM-1s-1) and [Mn(1,4-DO2AMBz)]2+ (r1p=3.5 mM-1s-1) and that of
free Mn2+ (r1p=8.0 mM-1s-1). The longitudinal relaxation times were
measured by the inversion recovery method (180° - τ- 90°) by using
8 different τ values. The measurements were performed with 1.0
mM [Mn(1,4-BzDO2AM)]2+ and [Mn(1,4-DO2AMBz)]2+ solutions in
the presence of a 10 to 20-fold excess of Zn2+. The temperature was
maintained at 25 °C and the ionic strength of the solutions was kept
constant (0.1 M KCl). For keeping the pH values constant, N-
methylpiperazine (pH range of 4.1 – 5.2) and piperazine (pH range
of 4.7 – 6.6) buffers (0.01 M) were used. The pseudo-first-order rate
constants (kd) were calculated with the use of the Eq. (9)
Acknowledgements
L.T. and M.B. are grateful to UNIUPO for support. A. F. thanks
MIUR (Italy) for a PhD grant. Authors C. P.-I. and D. E.-G. thank
Ministerio de Economía y Competitivad (CTQ2015-71211-REDT and
CTQ2016-76756-P) for generous financial support and Centro de
Supercomputación de Galicia (CESGA) for providing the computer
facilities.
d t
R1t = (R10 − R1e )e−k + R1e
(9)
t
e
where R10, R1 , and R1 are the relaxation rate (1/T1) values at the
References
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Table of Contents
The Mn2+ complexes of two differently benzyl-substituted 1,4-DO2AM ligands show a strong increase in
relaxivity upon Human Serum Albumin binding