Manganese Complex of a Rigidified 15-Membered Macrocycle: A Comprehensive Study

Article abstract of DOI:10.1021/acs.inorgchem.0c01053

Owing to the increasing importance of manganese(II) complexes in the field of magnetic resonance imaging (MRI), large efforts have been devoted to find an appropriate ligand for Mn(II) ion encapsulation by providing balance between the seemingly contradictory requirements (i.e., thermodynamic stability and kinetic inertness vs low ligand denticity enabling water molecule(s) to be coordinated in its metal center). Among these ligands, a large number of pyridine or pyridol based open-chain and macrocyclic chelators have been investigated so far. As a next step in the development of these chelators, 15-pyN3O2Ph and its transition metal complexes were synthesized and characterized using established methods. The 15-pyN3O2Ph ligand incorporates both pyridine and ortho-phenylene units to decrease ligand flexibility. The thermodynamic properties, protonation and stability constants, were determined using pH-potentiometry; the solid-state structures of two protonation states of the free ligand and its manganese complex were obtained by single crystal X-ray diffractometry. The results show a seven-coordinate metal center with two water molecules in the first coordination sphere. The longitudinal relaxivity of [Mn(15-pyN3O2Ph)]2+ was found to be 5.16 mM-1 s-1 at 0.49 T (298 K). Furthermore, the r2p value of 11.72 mM-1 s-1 (0.49 T), which is doubled at 1.41 T field, suggests that design of this Mn(II) complex does achieve some characteristics required for contrast imaging. In addition, 17O NMR measurements were performed in order to access the microscopic parameters governing this key feature (e.g., water exchange rate). Finally, manganese complexes of ligands with analogous polyaza macrocyclic scaffold have been investigated as low molecular weight Mn(CAT) mimics. Here, we report the H2O2 disproportionation study of [Mn(15-pyN3O2Ph)]2+ to demonstrate the versatility of this ligand scaffold as well.

Full text of DOI:10.1021/acs.inorgchem.0c01053

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Article
Manganese Complex of a Rigidified 15-Membered Macrocycle: A
Comprehensive Study
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Kristof Pota, Eniko Molnar, Ferenc Krisztian Kalman, David M. Freire, Gyula Tircso,*
and Kayla N. Green*
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ABSTRACT: Owing to the increasing importance of manganese(II)
complexes in the field of magnetic resonance imaging (MRI), large efforts
have been devoted to find an appropriate ligand for Mn(II) ion
encapsulation by providing balance between the seemingly contradictory
requirements (i.e., thermodynamic stability and kinetic inertness vs low
ligand denticity enabling water molecule(s) to be coordinated in its metal
center). Among these ligands, a large number of pyridine or pyridol based
open-chain and macrocyclic chelators have been investigated so far. As a
next step in the development of these chelators, 15-pyN3O2Ph and its
transition metal complexes were synthesized and characterized using
established methods. The 15-pyN3O2Ph ligand incorporates both pyridine
and ortho-phenylene units to decrease ligand flexibility. The thermody-
namic properties, protonation and stability constants, were determined using pH-potentiometry; the solid-state structures of two
protonation states of the free ligand and its manganese complex were obtained by single crystal X-ray diffractometry. The results
show a seven-coordinate metal center with two water molecules in the first coordination sphere. The longitudinal relaxivity of
[Mn(15-pyN3O2Ph)]2+ was found to be 5.16 mM−1 s−1 at 0.49 T (298 K). Furthermore, the r2p value of 11.72 mM−1 s−1 (0.49 T),
which is doubled at 1.41 T field, suggests that design of this Mn(II) complex does achieve some characteristics required for contrast
imaging. In addition, 17O NMR measurements were performed in order to access the microscopic parameters governing this key
feature (e.g., water exchange rate). Finally, manganese complexes of ligands with analogous polyaza macrocyclic scaffold have been
investigated as low molecular weight Mn(CAT) mimics. Here, we report the H2O2 disproportionation study of [Mn(15-
pyN3O2Ph)]2+ to demonstrate the versatility of this ligand scaffold as well.
potential free gadolinium toxicity.14−16 It is now proven that
the injected dose is not completely excreted, which can lead to
INTRODUCTION
■
As first shown by Pedersen, 15-membered macrocycles
delayed toxicity. The first association between MRI agents and
containing five heteroatoms have the ability to function as
a disease was made in 2006, when patients with impaired renal
complexing agents.1 From this starting point, a large library of
function were diagnosed with nephrogenic systemic fibrosis
ligands bearing oxygen and nitrogen donor atoms have been
(NSF).17,18 More recently, a series of reports revealed long-
synthesized and explored for a diverse field of applications.2−4
term gadolinium retention in the central nervous system in the
In these studies, crown- and aza-crown ethers have shown the
case of patients without renal impairment.19
capability to act as phase-transfer catalysts in organic
Manganese complexes have been proposed as possible
reactions.5−8 Mn(II) complexes of these ligands are known
alternatives for the GBCAs. The d5 Mn(II) ion is a very
as highly active and stable superoxide dismutase (MnSOD) or
effective relaxation agent due to its preference for a high-spin
catalase (Mn(CAT)) mimics.9−11 Another promising applica-
(S = 5/2) electron configuration and a characteristically long
tion is the use of the high-spin Mn(II) chelates as magnetic
longitudinal electron spin relaxation time.20 However, the
resonance imaging (MRI) contrast agents (CAs).12
coordination number is limited to six or seven in aqueous
At present, about 40% of all MRI procedures use one of the
seven Gd(III)-based contrast agents (GBCAs) that are
Received: April 9, 2020
commercially marketed and available in the U.S., Europe, or
Japan to diagnose tissue and vascular abnormalities.13
However, it is important to note that the use of some of the
agents containing a linear ligand are suspended or restricted in
Europe, and warnings have been issued by the United States
Food and Drug Administration (FDA) due to concerns about
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Figure 1. Structure of ligands studied or discussed in the present work.
chelates with Mn(II), compared to the Gd(III) coordination
number of nine, a typical value for later lanthanide metal ions.
For the targeted imaging activity, the Mn(II) complexes must
accommodate at least one water molecule in the first
coordination sphere. Hence, hexadentate, open-chain ligands
garnered increased interest in recent years as a means of
replacing GBCAs.21,22
Accordingly, the objective of the study was to synthesize and
characterize the 15-pyN3O2Ph ligand and its transition metal
complexes, with an emphasis on the Mn(II) complex (Figure
1). Here, we report the synthesis and crystal structures of the
free ligand and its manganese bound form. The thermody-
namic properties, protonation, and stability constants of 15-
pyN3O2Ph and several of its alkaline-earth and transition metal
complexes were determined by pH-potentiometric titrations
using 0.15 M NaCl ionic strength to model physiological
conditions. Furthermore, relaxation properties of the complex
Several studies have shown that ligand rigidity may be a key
feature to maintain an intact complex upon injection for
imaging in vivo.22−25 It was shown that the rigidification of the
backbone leads to a 150−250 fold increase in the kinetic
inertness of the Mn(II) complexes of the trans-CDTA and
PhDTA ligands compared to the EDTA congener (Figure
1).21,26 Inclusion of a pyridine moiety into a macrocycle is an
alternative approach to increase the resistance of these chelates
toward proton assisted dissociation. It was shown that the
dissociation half-life of the [Mn(PCTA)]− complex (pH = 7.4)
is more than 5 times higher compared to the [Mn(DO3A)]−.27
The validity of this second method has already been
successfully reported for 15-membered pentaaza-macrocycles:
the [Mn(15-pyN5)]2+ complex should remain intact during the
course of an MRI experiment under physiological conditions.12
On the basis of these reports, we set out to further decrease
the flexibility of pentadentate chelators using the 15-membered
macrocyclic base structure (15-aneN3O2) with the inclusion of
both the pyridine (15-pyN3O2) and the ortho-phenylene units.
1
formed with Mn(II) were studied by H relaxometry, while
17O NMR studies were used to gain information about the
exchange rate of the metal ion bound solvent molecules.
Manganese complexes of 15-membered pentaaza-crown
ether macrocycles have also been extensively explored as
mimics for metalloenzymes with a particular focus on catalase
reactivity.11 Catalase enzymes are essential for living cells to
protect them against oxidative stress. Their main function is to
catalyze the disproportionation of a toxic oxygen metabolite,
hydrogen peroxide (H2O2), into molecular oxygen (O2) and
water (H2O) before it is transformed into reactive oxygen
species (ROS) like the hydroxyl radical (OH•) through other
redox pathways.28 Most catalase enzymes are composed of an
iron-protoporphyrin IX prosthetic group, but some living
organisms utilize manganese based enzymes (e.g., Mn(CAT))
instead. Ligand features modulate the stability and activity of
B
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a
Scheme 1. Synthesis of 15-pyN3O2Ph
a
Conditions: (i) Boc2O, Et3N, CH2Cl2, RT, 18 h. (ii) K2CO3, MeCN, reflux, 24 h. (iii) Step 1: AcCl, MeOH, RT, 24 h. Step 2: NaOH(s), MeCN.
(iv) MnO2, CHCl3, reflux, 5 h. (v) Step 1: MnCl2, MeOH, reflux, 2 h. Step 2: NaBH4, RT, 14 h. Step 3: H2O on air, RT, 1 h.
(100.6 MHz, CDCl3): δ 158.4, 149.0, 136.7, 121.1, 120.4, 113.3, 68.4,
53.9, 49.0. HRMS (ESI, m/z) calcd for C17H22N3O2 [M + H]+:
300.1712. Found: 300.1901; calcd for C17H21N3NaO2 [M + Na]+:
322.1531. Found: 322.1585. Elemental analysis for 2C17H21N3O2·
2C2H5OH·HCl found (calcd): C, 62.23 (62.75); H, 7.40 (7.62); N,
11.47 (11.55).
Crystal Structure Determination. Single crystals of 15-
pyN3O2Ph were obtained by slow evaporation of a CH3OH solution.
Low quality single crystals of the perchlorate salt of 15-pyN3O2Ph
were obtained by slow evaporation of the aqueous ligand solution,
which was acidified by two drops of perchloric acid (70%). The
manganese complex solution was prepared by mixing the aqueous
solutions of the ligand (cL = 17 mM) and manganese(II) chloride
(cMn = 50 mM) in a Mn/L molar ratio of 1:1.02. The solution was
evaporated under reduced pressure, and the residue was dissolved in
EtOH and filtered through a syringe filter (0.45 μm PTFE
membrane). Colorless single crystals suitable for the X-ray diffraction
analysis were prepared by slow evaporation of the EtOH solution at
room temperature.
A Leica MZ 75 microscope was used to identify samples suitable
for analysis. A Bruker APEX-II CCD diffractometer was employed for
crystal screening, unit cell determination, and data collection, which
were obtained at 100 K. The Bruker D8 goniometer was controlled
using the APEX3 software suite.35 The samples were optically
centered with the aid of a video camera so that no translations were
observed as the crystal was rotated through all positions. The X-ray
radiation employed was generated from a Mo Kα sealed X-ray tube (λ
= 0.71076 Å) with a potential of 50 kV and a current of 30 mA, fitted
with a graphite monochromator in the parallel mode (175 mm
collimator with 0.5 mm pinholes).
Potentiometric Measurements. The MgCl2, CaCl2, MnCl2,
FeCl2, CuCl2, and ZnCl2 stock solutions were prepared from
analytical grade chemicals, and their concentrations were determined
by complexometric titrations with standardized Na2H2EDTA and an
eriochrome black-T (EBT) indicator for MgCl2 and CaCl2, EBT
indicator in the presence of ascorbic acid and triethanol-amine for
MnCl2, thiosalicylic acid indicator in the presence of ascorbic acid and
pyridine for FeCl2, PAR indicator in the presence of NH4OAc for
CuCl2, and EBT indicator in the presence pH 10 NH3/NH4Cl buffer
for ZnCl2. The concentration of the ligand stock solution was
determined by pH-potentiometric titration. The protonation con-
stants were calculated with the following setup: 0.2 M carbonate free
NaOH titrant with 2 mM ligand solution at an initial volume of 6 mL.
Titrations were performed at 25.0 0.1 °C and an ionic strength of I
= 0.15 M NaCl to model the background present in body fluids. An
inert atmosphere was provided by a constant passage of dry N2
through the sample. The protonation constants of the ligand (log KiH)
are defined as
the manganese center toward hydrogen peroxide disproportio-
nation; in particular aza-oxa-macrocyclic complexes of
manganese have been studied as Mn(CAT) models due to
their high formation constants. Structural changes in the
substituents of the macrocycle seem to have an effect in both
the kinetic of the reaction and mechanism pathway, although
the latter is still unclear.11 The [Mn(15-pyN3O2Ph)]2+
complex was found to catalyze the disproportionation of
H2O2. To quantify its activity, a series experiments was
conducted under controlled conditions.
EXPERIMENTAL SECTION
■
General. The pyridine-2,6-dicarbaldehyde (1),12,29−31 tert-butyl-
(2-bromoethyl)carbamate (2),32 di-tert-butyl-([1,2-phenylenebis-
(oxy)]bis[ethane-2,1-diyl])dicarbamate (3),33 and 2,2′-[1,2-
phenylenebis(oxy)]diethanamine (4;34 Scheme 1) were prepared
according to literature procedures (or slightly modified, see
Supporting Information). Methanol was distilled from KOH and
stored over 4 Å molecular sieves. Other solvents and chemicals were
purchased from commercial sources and used as received. NMR
spectra were recorded on a Bruker Avance III 400 MHz High
1
Performance Digital NMR Spectrometer at 298 K: H (400.1 MHz),
TMS (internal): δ 0.00 ppm; 13C (100.6 MHz), TMS (internal): δ
0.00 ppm. Multiplicity of the signals is indicated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Deuterated
solvents (Aldrich: CDCl3, 99.8% D; Cambridge Isotope Laboratories:
CD3OD, 99.8% D; DMSO-d6, 99.96% D) were used as received. Mass
spectra were obtained on an Agilent 1200 series 6224 TOF LC/MS
spectrometer equipped with an electro-spray ion source at 75 V
(positive ion mode).
Synthesis of Cyclo(2,6-pyridinediylmethyleneiminoethyle-
neoxy-1,2-phenyleneoxyethyleneiminomethylene) (15-py-
N3O2Ph). Compound 4 (0.476 g, 3.52 mmol) and MnCl2·4H2O
(0.697 g, 3.52 mmol) were dissolved in dry MeOH (50 mL). A
solution of 1 (0.691 g, 3.52 mmol) in dry MeOH (30 mL) was added
dropwise over 15 min. The reaction mixture was refluxed for 2 h.
After cooling to 0 °C, NaBH4 (1.81 g, 47.8 mmol) was added in small
portions. The solution was left stirring overnight at RT. Water (40
mL) was slowly added, which induced the precipitation of Mn(OH)2
and immediate oxidation. The resulting suspension was stirred at RT
for 1 h. MeOH was removed in vacuo, and the remaining aqueous
phase was filtered on a frit under a vacuum. The brown precipitate,
which formed upon quenching, was washed with CH2Cl2 (3 × 40
mL), and the aqueous phase was extracted with the CH2Cl2. The
organic layer was dried with anhydrous Na2SO4, and the solvent was
evaporated under reduced pressure. The crude product was purified
by column chromatography (SiO2, EtOH/cc·NH3(aq.) = 100:1) and
dried in vacuo at 50 °C. Yield: 0.342 g (33%), light yellow solid, Rf =
0.43 (EtOH/cc·NH3(aq.) = 100:1). 1H NMR (400 MHz, CDCl3): δ
7.56 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.6 Hz, 2H), 6.90 (s, 4H), 4.16
(t, J = 4.8 Hz, 4H), 3.95 (s, 4H), 3.05 (t, J = 4.8 Hz, 4H). 13C NMR
i+
[HiL ]
KiH
=
[Hi−1L
(i−1)+][H+]
(1)
C
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where i = 1, 2, and 3 and [Hi−1L(i−1)+] and [H+] are the equilibrium
concentrations of the ligand in different protonation states and
hydrogen ions, respectively. The potentiometric titrations were
carried out with a Metrohm 785 DMP Titrino workstation and a
Metrohm 6.0234.100 combined electrode in the pH range of 2.0−
11.8. For the calibration of the microelectrode, KH-phtalate (pH =
4.008)36 and borate (pH = 9.177)37 buffer standards were used, and
the [H+] were calculated from the measured pH values by applying
the method proposed by Irving et al.38 A solution of approximately
0.01 M HCl was titrated with 0.2 M NaOH solution (I = 0.15 M
NaCl), and the differences between the measured and calculated pH
values (pH < 2.2) were used to calculate the [H+] from the pH values
measured in the ligand titrations. The experimental points above pH
11 for the acid−base titration were utilized to determine the ionic
product of water (13.840) in case of our experimental setup.
PSEQUAD software was used to calculate the equilibrium
constants.39 The protonation constants of the 15-pyN3O2Ph ligand
were determined by titrating the ligand solution (acidified with a
known volume of standard HCl solution) with 0.2 M NaOH at 0.15
M NaCl ionic strength in the 2.0−11.8 pH range. The log KiH values
were calculated from 150 V (mL)−pH data pairs. To determine the
stability constants of the complexes formed with different metal ions,
potentiometric titrations were carried out at a 1:1 metal-to-ligand
molar ratio, with 2% ligand excess to prevent the hydrolysis of the
metal ions, allowing 1 min for the equilibration to occur (the number
of data pairs were between 70 and 160).
water (10% H217O, NUKEM Isotopes Imaging GmbH) was added to
the solutions to reach a 2% enrichment. The fit of the 17O NMR data
was performed using Micromath Scientist calculation program via
least-squares fitting procedure.
Reaction of Mn(II) Complex with H2O2 and Quantification
of O2 Evolution. The reactions were performed in a sealed pressure
vessel (15 mL) equipped with a stirring bar and a three-way manifold
valve. Two of the lines (PTFE tubing) were connected to the cell: one
was used for N2 and the other to inject the aliquot of H2O2. The
pressure vessel was sealed using a PEEK bushing connected to
threaded fittings (IDEX Health and Science) for custom sealing
between the gas tubing, microsensor and reaction vessel. The pressure
of the vessel was kept at 1 atm using a snorkel line with minimal loss
of headspace during the catalytic studies. The sensor was calibrated
using N2 and air (0 and 159 mmHg) under atmospheric pressure.
The metal complex was prepared in aqueous solution at pH = 8
using stock solutions of 15-pyN3O2Ph, buffer, and MnCl2. At pH 8,
95% of the Mn(II) ions are chelated by the ligand. Tris-
(hydroxymethyl)aminomethane buffer was used to maintain the
desired pH during the experiments. The metal complex was prepared
by mixing the corresponding amount of the stock solution of ligand,
buffer, and MnCl2 in a vial to achieve the targeted concentration for
the H2O2 disproportionation studies ([15-pyN3O2Ph] = 2.04 mM;
[buffer] = 50 mM; [Mn(II)] = 2 mM). The vessel was loaded with
1.5 mL of the catalyst and pressurized with N2 gas prior to every
measurement. The electrode signal was read continuously and
measured every 0.2 s until a steady signal was obtained for 2 min,
then 0.5 mL of H2O2 solution was injected ([H2O2] = 600 mM,
1H-Relaxometric Studies. Measurements of longitudinal (T1)
and transverse (T2) relaxation times were performed by using Bruker
Minispec MQ-20 and MQ-60 NMR Analyzers working at 0.49 T
(corresponding to 20 MHz proton Larmor frequency) and 1.41 T
(corresponding to 60 MHz proton Larmor frequency), respectively.
The temperature of the sample holder was set (25.0 0.2 °C) and
controlled with the use of a circulating water bath. The T1 values were
determined with the inversion recovery method (180x° − τ − 90x°)
by averaging four to six data points obtained at 10 different τ delay
values. The transverse relaxation times (T2) were measured by using
the Carr−Purcell−Meiboom−Gill sequence (CPMG) by averaging
again four to six identical data points.40 The r1p and r2p relaxivities of
the complex were determined by using batch samples (lying in the
concentration range of 0.5−2.0 mM) prepared under a nitrogen
atmosphere having the ligand present at a 1.8-fold excess in the
presence of 5 equiv of a hydroxyl-amine reducing agent (to prevent
the oxidation of the Mn(II)). The pH in these samples was kept
constant at pH = 8.14 with the use of HEPES buffer (I = 0.15 M
NaCl, T = 298 K). By relying on the species distribution curves under
these conditions, only one Mn(II) ion containing species is present in
solution, which is the [Mn(15-pyN3O2Ph)]2+ complex. To confirm
the complex formation and its pH range, a sample containing 1 mM
of the ligand and Mn(II) (Vtot = 6 mL) was titrated with NaOH under
a N2 protected atmosphere, and the 1/T1,2p data were collected in the
pH range of 4.5−11.0.
[buffer] = 50 mM). The signal was recorded as ΔPO vs time until a
2
plateau was achieved. The initial concentrations of the species before
starting the reaction were [15-pyN3O2Ph] = 1.54 mM, [Mn(II)] = 1.5
mM, and [H2O2] = 150 mM.
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of 15-pyN3O2Ph was achieved by
convergent pathways (Scheme 1). The pyridine containing
unit of 15-pyN3O2Ph, pyridine-2,6-dicarbaldehyde (1), was
prepared from the commercially available 2,6-bis-
(hydroxymethyl)pyridine through partial oxidation of the
hydroxyl groups with MnO2 according to literature proce-
dures.12,29−31 The other half of the macrocycle was prepared in
a three step synthesis, which started with the protection of the
amine functional group of commercially available 2-bromoe-
thylamine as the carbamate. The Boc protecting group was
chosen because of its increased resistance to basic and
nucleophile reagents as well as its ease of removal with close
to quantitative yields.34 The reagent ratio was modified in the
case of the amine protection to remove the excess Boc2O from
the reaction mixture. The alkylation of the catechol was based
on the literature procedure.33 The workup of this reaction was
modified, and as a result, we were able to separate the pure
mono- and bis-alkylated products utilizing the difference
between their solubility in water at different pH values. The
deprotection step was achieved with excellent yield (94%), but
the free base form of the amine was necessary for the
cyclization, which led to a slightly decreased yield of 78%.
Mn(II) ions were used for cyclization of 1 and 4 in a one-pot
template synthesis; the reaction conditions to prepare the
intermediate Schiff-base and subsequent reduction paralleled
those described in previously published procedures for other
triaza-dioxa macrocyclic systems (Scheme 1).12,43 The final
product was obtained in a 33% overall yield as a light-yellow
solid after purification by column chromatography, and
17O NMR Studies. Longitudinal (1/T1) and transverse (1/T2)
relaxation rates and chemical shifts of an aqueous solution of the
Mn(II) complex (pH = 8.14, at 1.9 mM concentration) and of a
diamagnetic reference (HClO4 acidified water, pH = 3.3) were
measured in the temperature range 273−348 K using a Bruker Avance
400 (9.4 T, 54.2 MHz) spectrometer. Analogously to the 1H
relaxometric measurements, the ligand was used in a 100% excess
compared to Mn(II) to limit the free metal ion concentration. In
order to avoid the oxidation of the Mn(II) ion in the complex,
hydroxyl−amine was added to the sample (at 5 mM concentration),
based on accepted protocols. The temperature was determined
according to previous calibration routines by means of ethylene glycol
as a standard.41 1/T1 and 1/T2 values were determined by the
inversion−recovery and the Carr−Purcell−Meiboom−Gill spin−echo
technique, respectively.40 The 17O NMR technique for accessing
water exchange rates has been described previously.42 To avoid
susceptibility corrections of the chemical shifts, a glass sphere fitted
into a 10 mm NMR tube was used during the measurements. To
increase the sensitivity of 17O NMR measurements, 17O enriched
1
connectivity was confirmed by H and 13C NMR, MS, and
elemental analysis.
D
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Solid State Structures: Crystal Structure of the
Ligand. Two different protonation states of the 15-pyN3O2Ph
ligand were characterized by single crystal X-ray diffractometry.
The base form ([15-pyN3O2Ph]0, Figure 2) possesses a near
Solid State Structure of the Mn(II) Complex. The
manganese complex was prepared by the addition of MnCl2 to
an aqueous solution of 15-pyN3O2Ph. Materials suitable for X-
ray diffraction analysis were obtained by evaporating MeOH
solutions of the crude product. The solid state model has a
composition of [Mn(15-pyN3O2Ph)(H2O)2]Cl2; a Mn(II) ion
can be found in the center of the planar macrocyclic cavity
with three nitrogen and two oxygen atoms in the equatorial
plane and two water molecules in the apical positions forming
the pentagonal-bipyramidal coordination sphere (Figure 4). A
Figure 2. Molecular structure of 15-pyN3O2Ph (front and side view).
The thermal ellipsoids are drawn with 50% probability.
planar structure with a pseudo-C2 symmetry axis passing
through carbon C16, nitrogen N1, and the center of the
benzene ring (bonds C5−C10, C7−C8). A similar but more
emphasized symmetry element can be observed in the acid
form (propeller like twist, best visualized from the top view,
[H3(15-pyN3O2Ph)]3+, Figure 3), which is analogous to the
structure of the parent molecule ([H3(15-pyN3O2)]3+).12 The
proton on the pyridine nitrogen atom (N1) is hydrogen
bonding with oxygen atoms O1 and O2 resulting in dN−O ∼ 2.8
Å, which represent a drastic shrink in the macrocyclic cavity
compared to the base form, where the same distances are
around 4.2 Å. This structural change can help explain the
observed nearly instantaneous dissociation of the Mn(II)
complex upon acidification.
Figure 4. Molecular structure of the [Mn(15-pyN3O2Ph)(H2O)2]2+
unit (front and side view). The thermal ellipsoids are drawn with 50%
probability.
seven coordinate Mn(II) ion has been observed with amino-
polycarboxylate ligands like EDTA, but this is only the second
reported crystal structure with two water molecules in the first
coordination sphere with a pentadentate macrocycle.44,45 The
Mn(II) complex of 15-pyN3O2 and 15-pyN5 had two chlorides
or one chloride and one water molecule, respectively.12 Of
course, these chlorides are replaced by water molecules in
Figure 3. Molecular structure of [H3(15-pyN3O2Ph)]3+ (front, top, and side view). Hydrogen atoms, solvent molecules, and counterions are
omitted for clarity. The thermal ellipsoids are drawn with 50% probability.
E
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aqueous solution based on the 17O NMR experiments
described below. The previously mentioned π−π stacking of
the pyridine and phenylene moieties can be observed in the
unit cell of the manganese complex, similarly to the free base
form of the ligand. The only major difference is that the C2
symmetry of the free base form was lost because the metal
center forced the two secondary amines to reside on the same
side of the plane determined by the pyridine ring (Figures 2
and 3). Four out of the five chelate rings in the complex have
alternating δ/λ conformations starting from the pyridine
nitrogen atom (δλ−δλ). The only exception is the five-
membered ring opposite the pyridine, which is planar due to
structural requirements of the phenylene group. All coordina-
tion bonds are within the expected range and are consistent
with those reported in related structures (Table 1).10,12,45−47
and 3). For these experiments, 0.15 M NaCl ionic strength was
used to model the conditions present in bodily fluids. Two
Table 3. Stability Constants (log K) of Complexes Formed
with Divalent Metal Ions and 15-Membered Triaza-Dioxa
Macrocycles (T = 298 K, I = 0.15 M NaCl, Charges in the
Equilibrium Quotients Were Omitted for Clarity)
15-
15-
d
equilibrium quotient
Mg(II) [ML]/([M][L])
15-aneN3O2 pyN3O2 pyN3O2Ph
1.84(9)
[ML]/([M(OH)L]
[H])
Ca(II)
[ML]/([M][L])
2.04
1.85(8)
[ML]/([M(OH)L]
[H])
Mn(II) [ML]/([M][L])
11.92
a
a
a
6.63
7.18
11.69
5.62(3)
10.50(4)
[ML]/([M(OH)L]
[H])
[ML]/([M][L])
[ML]/([M(OH)L]
[H])
[ML]/([M][L])
[ML]/([M(OH)L]
[H])
[ML]/([M][L])
[ML]/([M(OH)L]
[H])
Table 1. Selected Interatomic Distances (Å) Found in the
Crystal Structure of [Mn(15-pyN3O2Ph)(H2O)2]Cl2
Compared to Representative Examples of Mn(II)-
Complexes with Pentadentate Macrocycles
Fe(II)
Co(II)
Cu(II)
7.79
7.35(3)
10.78(5)
a
9.1
8.49
9.48
11.80
7.65(4)
[Mn(15-
[Mn(15-
[Mn(15-
pyN3O2)
[Mn(15-
pyN3O2Ph)
(H2O)2]2+
aneN5)
Me2pyN )
5 b
a
c
Cl2]
(H2O)2]2+
(H2O)Cl]+
a
b
15.72, 15.27
13.91
8.34
13.97(2)
8.95(6)
Mn−N1
Mn−N2
Mn−N3
Mn−O1
Mn−O2
Mn−O1w
2.26
2.41
2.45
2.278
2.343
2.352
2.229
2.334
2.300
2.250
2.305
2.230
2.340(4)
2.346(4)
2.339(4)
2.167(3)
2.208(3)
2.207(13)
2.141(16)
a
b
8.87,
−
[M(OH)L]/
12.57
8.58
([M(OH)2L][H])
a
b
Zn(II)
[ML]/([M][L])
8.95, 8.85,
7.70(3)
9.62(5)
c
8.91
2.282
2.241
a b
c
,
[ML]/([M(OH)L]
[H])
−, 8.85
10.31
Mn−O2w
a
b
c
Ref 46. Ref 45. Ref 12.
a
b
c
Ref 49 (I = M KNO3). Ref 48 (I = 0.1 M NaNO3). Ref 51 (I = 0.1
d
M NaCl). Ref 12 (I = 0.1 M Me4NCl).
The Mn−N distances (2.34−2.35 Å) of [Mn(15-
pyN3O2Ph)(H2O)2]Cl2 are slightly longer than the Mn−O
bonds (2.14−2.21 Å) with either the oxygen atoms in the
macrocycle or the water molecules. The bond angles between
the neighboring donor atoms of the macrocycle and the central
Mn(II) ion range from 70.6 to 74.5°, which is consistent with
the theoretical value of 72° of the regular pentagon. Also, the
regular trans-apical coordination is confirmed by the bond
angle of 174.9° between the water molecules and the divalent
center (Table S5). The Mn−O distance for the coordinated
water molecules is slightly shorter than in complexes with
similar pentadentate macrocycles (Table 1). A possible
explanation for this result might be the increased planarity of
the ligand, which is induced by the two aromatic systems on
the opposite sides of the structure.
protonation events were found for 15-pyN3O2Ph that are
attributed to subsequent protonation of the secondary amine
nitrogen atoms. However, protonation of the pyridine nitrogen
atom is outside of the limits of our experimental conditions.
These results are in good agreement with previously reported
values for 15-membered triaza-dioxa macrocycles.12,48,49
Comparison of the stepwise log KiH values (Table 2) shows
that, with the introduction of the pyridine moiety, the basicity
of the adjacent secondary amine nitrogen atoms decreased
only slightly (approximately 0.5 log units) due to the electron-
withdrawing effect of the aromatic system compared to 15-
aneN3O2. However, the total basicity decreased noticeably
because of the replacement of the amine nitrogen atoms with a
pyridine unit. On the other hand, only negligible attenuation of
the log KiH values and the basicity of the ligand are observed
between 15-pyN3O2 and 15-pyN3O2Ph after the introduction
of the phenylene group on the opposite side of the molecule.
This difference is attributed to the electron withdrawing nature
of the phenylene group compared to ethylene. The species
distribution diagram for the 15-pyN3O2Ph can be found in the
Supporting Information (Figure S17).
Equilibrium Studies. Stepwise protonation constants of
15-pyN3O2Ph as well as stability constants of its complexes
with various alkaline earth and transition metal ions were
determined by standard pH-potentiometric titrations (Tables 2
Table 2. Stepwise Protonation Constants of 15-Membered
Triaza-Dioxa Macrocycles (T = 298 K, I = 0.15 M NaCl)
c
15-aneN3O2
15-pyN3O2
15-pyN3O2Ph
a
b
b
b
H
H
H
The values of the stability constants for the complexes of 15-
pyN3O2Ph with a range of divalent alkaline-earth and first-row
transition metal ions (Table 3) were determined using the
same conditions as for the ligand titrations. The results show
that 1:1 complexes form for each of the systems studied. The
stability of the ternary hydroxo chelates could be calculated for
almost all metal ions. The Irving−Williams’ order of stability is
log K1
log K2
log K3
9.29, 9.51
8.82
7.80
8.53(3)
7.63(3)
a
8.50, 8.47
a
2.12, 2.30
a
b
Σ logKiH
19.91, 20.28
16.62
16.16
a
b
c
Ref 48 (I = 0.1 M NaNO3). Ref 49 (I = 0.1 M KNO3). Ref 12 (I =
0.1 M Me4NCl).
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followed for all the transition metal ions with a maximum value
for the Cu(II) complex.50
plotted together as a function of pH in the graph depicted in
Figure 6. The comparison of the curves indicates that the
A few important conclusions can be drawn from the
comparison of the formation constants for the three 15-
membered aza-oxa macrocycles (Figure 5). First, the effect of
Figure 6. Species distribution diagram of the complex [Mn(15-
pyN3O2Ph)]2+ calculated in the pH range of 4 to 11 (by using 1.0
mM of Mn(II) and 2.0 mM ligand; solid lines) and the plots of the 1/
T1p (blue diamonds) and 1/T2p (green circles) values measured at
1.41 T and 0.15 M NaCl at 298 K (charges are omitted for clarity).
Figure 5. Transition metal complex stabilities of the discussed ligands.
equilibrium model applied for the fitting of pH-potentiometric
data describes the complex formation correctly. Formation of
[Mn(15-pyN3O2Ph)]2+ starts only at pH 5.70, and it reaches a
maximum only near pH 8.0, even with ligand excess applied.
However, raising the pH above 9.0 results in the formation of
ternary hydroxo species. These results are consistent with the
formation of a relatively weak Mn(II) complex with the 15-
pyN3O2Ph ligand, partially due to an excess of ligand rigidity.
On the basis of the information presented in Figure 6, the
relaxivity of the [Mn(15-pyN3O2Ph)]2+ complex was deter-
mined by plotting the 1/T1p values of samples as a function of
complex concentration (Figure S20) at pH 8.14 (set by
HEPES buffer). The relaxivity values were compared to data
measured for structurally similar systems under the same
conditions (Table 4). The relaxivity of the [Mn(15-
pyN3O2Ph)]2+ complex (5.16 mM−1 s−1 at 0.49 T (298 K))
is in the same range as those determined for 15-pyNxO5−x
derivatives.12 This value is in agreement with the presence of
more than one water molecule in the inner sphere of the
Mn(II) center, which was validated by 17O NMR following the
method proposed by Gale et al., as expected based on an
the pyridine moiety (15-pyN3O2) on the stability of Mn(II)
and Co(II) complexes is overturned with the introduction of
the second aromatic system to the macrocyclic framework (15-
pyN3O2Ph). The respective constants are 1.56 and 1.83 log
units lower for the chelates formed with 15-pyN3O2Ph. This
behavior is most likely due to the highly increased rigidity of
the ligand, which causes a decrease in the entropy component
of the binding configurational entropy of the ligand. However,
in case of the Cu(II) binding affinity this structural
modification is not present, the value describing the strength
of interaction is almost within the error of the experimental
technique (for the species distribution diagram, see Figure
S18). Unfortunately, the rather small stability constant for the
Mn(II) complex means that even by pH 8, only 95% of the
cation is chelated by the macrocycle (Figure S19). Therefore,
the addition of the phenylene moiety has decreased the
formation stability of the Mn(II) outside of the window of
applicability for imaging studies, thus it represents a stopping
point for consideration in future ligand design.
1H-Relaxometric Studies. Complex formation involving
paramagnetic metal ions can be studied through 1H
relaxometric method by following the longitudinal (T1) or
transverse (T2) relaxation times (or r1p and r2p relaxivities) as a
function of pH. The data obtained by the given technique
might provide supporting data to the equilibrium model
established (often set by the expert) for the fitting of the pH-
potentiometric data by comparing the species distribution
curves calculated using stability constants determined by pH-
potentiometry with the pH profile of the relaxivity. Such a
profile was first obtained for a sample containing equimolar
amounts of the metal ion and the ligand (Figure S19). While
the given profile confirmed the complex formation in the
Mn(II)−15-pyN3O2Ph−H+ system, formation of the precip-
itate near pH = 9.5 (corresponding to the precipitation of
uncomplexed Mn(II)) narrows the pH-range available for the
study. Thus, we performed a titration of the sample with an
excess of the ligand to push the reaction toward full
complexation. Hydroxyl-amine was added to the sample in
parallel to overcome the oxidation of Mn(II). The normalized
relaxivities and molar fraction of the species present in solution
was calculated by using the pH-potentiometric measurements
Table 4. Relaxivities and Best Fit Parameters Obtained from
the Analysis of 17O NMR Data for the Mn(II) Complexes of
15-pyN3O2Ph,15-pyN3O2,15-pyN5, ENOTA, and 4-HET-
CDTA
4-
15-
15-
pyN5
HET-
a
a
b
c
parameter
15-pyN3O2Ph pyN3O2
ENOTA CDTA
r1p/r2p (mM−1
5.16/11.72
4.48/− 3.56/−
3.39/−
4.87/−
s−1) at 0.49 T
d
(298 K)
kex (107 s−1
ΔH‡ (kJ mol−1
)
0.64 0.05
0.38
35.3
−1
6.9
37.7
+32
5.5
20.5
−28
17.6
36.2
+34
298
)
34
3
ΔS‡ (J
−1 10
mol−1K−1
AO/η (106
)
c
43 14
38.6
38.6
32.7
40
rad s−1
)
1/T1e (107
11
8
7
298
s−1
)
a
b
c
d
Ref 12. Ref 56. Ref 57. r1p and r2p are 4.49 and 25.69 mM−1 s−1 at
1.41 T (298 K).
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analogy of the Mn(II) complexes formed with the parent
15-pyN5 and 15-pyN3O2 chelators.52 The relaxivity of [Mn(15-
pyN3O2Ph)]2+ is larger than those reported for the FDA-
approved Gd(III)-based contrast agents (for example,
Dotarem in water, r1p = 3.4 mM−1 s−1 at 0.49 T (313 K)
and 2.9 mM−1 s−1 at 1.41 T (310 K)).53 It should also be
noted that the [Mn(15-pyN3O2Ph)]2+ complex possesses a r2p
value of 11.72 mM−1 s−1 (0.49 T), which is doubled at a 1.41
T field, suggesting that [Mn(15-pyN3O2Ph)]2+ can be an
efficient T2 shortening agent.
ca. 50% higher than that of the [Mn(15-pyN3O2)]2+ complex
and 1 order of magnitude lower than the value determined for
the [Mn(15-pyN5)]2+ chelate. Accepting the explanation given
by Drahos et al., the strength of the hydrogen bond formed
between the water proton and the O donors of the ligand
affects the rate of the water exchange.12 The incorporation of
the phenyl group into the macrocyclic backbone increases the
rigidity of the coordination cavity around the Mn(II) ion due
to its planar structure, which in turn has a significant impact on
the length and strength of the hydrogen bonds involving the
coordinated water molecules. This might be one of the
explanations for the decreased water exchange rate of the
complex and is in agreement with the results obtained by X-ray
crystallography, which indicates a slight shortening of the Mn−
Ow bonds in [Mn(15-pyN3O2Ph)]2+ in comparison with
[Mn(15-Me2pyN5(H2O)2]2+ and [Mn(15-pyN3O2)(H2O)-
Cl]+.12,45 The activation entropy for [Mn(15-pyN3O2Ph)]2+
is close to zero (ΔS‡ = −1 10 J mol−1K−1), indicating that
the water exchange very likely occurs via an interchange
mechanism. The six-coordinated Mn(II) complexes frequently
go through associatively activated water exchange, while the
seven-coordinate chelates often show a dissociatively activated
process.56,57 Similar behavior was observed for the [Mn(15-
pyN3O2)]2+ complex as well.
17O NMR Measurements. In order to gain information on
the water exchange rate (kex) of the Mn(II) complex, one of
the most important physicochemical parameters affecting the
relaxivity of a paramagnetic metal complex, temperature
dependent 17O NMR experiments were carried out. The
transverse and longitudinal relaxation rates as well as the
chemical shift of the 17O signal in the presence and absence of
the paramagnetic agent can provide information on the kex, the
rotational motion, and the electronic parameters, while the
chemical shift is correlated to the number of the water
molecules directly coordinated to the paramagnetic metal
center (q). The relaxation rates (1/T1 and 1/T2) and chemical
shifts (Δωr) were measured for an aqueous solution of
[Mn(15-pyN3O2Ph)]2+ and for a diamagnetic reference at 9.4
T. The T1 values showed a negligible difference between the
Mn(II) complex and the reference and thus were not included
in the calculations. The Swift−Connick equations were used,
by assuming a simple exponential behavior for the electron
spin relaxation, for the fitting of the 1/T2r values and chemical
shifts.54,55 The plots of the experimental data together with the
fitted curves are depicted in Figure 7. The water exchange rate,
Catalytic H2O2 Disproportionation. In order to inves-
tigate the effect of the 15-pyN3O2Ph ligand on this reactivity,
H2O2 disproportionation reactions were carried out using a
one-pot approach.58 The pH of the solution was selected based
on the speciation curve; at pH 8, 95% of the Mn(II) ions are
chelated by the ligand (Figure S19). An O2 microsensor probe
(Unisense, Denmark) inserted into a sealed pressure vessel,
previously flushed with N2, was used to continuously measure
the partial pressure of O2 (PO ) in mmHg every 0.2 s. Addition
2
of H2O2 resulted in oxygen evolution based on the formation
of bubbles in the solution and a significant increase in PO
2
measured by the sensor. O2 evolution plateaued at 60 min.
Control reactions were carried out with either the ligand or the
metal by itself, which showed a small O2 evolution signal using
the same conditions, validating the need for the metal complex
in solution for catalysis to occur.
The results were plotted as ΔPO (mmHg) vs time (min)
2
(Figure 8). The O2 pressure increased continuously after the
Figure 7. Reduced 17O transverse relaxation rates (squares) and
chemical shifts (triangles) measured for the [Mn(15-pyN3O2Ph)]2+
complex (cMn2+ = 1.90 mM and cLig = 3.80 mM at pH = 8.14). The
solid lines correspond to the best fit of the data.
kex298, its activation enthalpy, ΔH†, the hyperfine coupling
298
constant, AO/ℏ, and 1/T1e were calculated (the activation
energy of electron spin relaxation was fixed to 1 kJ/mol). The
hydration number was set to q = 2 based on analogy to the
results published by Drahos et al. for the complexes [Mn(15-
pyN3O2)]2+ and [Mn(15-pyN5)]2+.12 This assumption was
confirmed by the transverse relaxation rates of the 17O nucleus
in the presence of the paramagnetic Mn(II) complex (q = 1.6
0.2).52
The reduced transverse 17O relaxation rates are in the slow
water exchange regime at lower temperatures reaching a
maximum at around 341 K (Figure S21). The kex value
characterizing the water exchange of [Mn(15-pyN3O2Ph)]2+ is
Figure 8. O2 evolution from H2O2 disproportionation reaction
catalyzed by [Mn(15-pyN3O2Ph)]2+. Initial conditions: [Mn(II)] =
1.5 mM, [H2O2] = 150 mM, [buffer] = 50 mM.
H
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Article
injection of H2O2. The turnover frequency (TOF) was
calculated at different times from the injection of H2O2 at 1,
10, 20, and 60 min (Table 5). At 60 min, the TOF is lower
Details of synthesis, NMR spectra, crystallographic data,
equilibrium and relaxometric studies, and details related
to the 17O measurements (PDF)
Accession Codes
Table 5. ΔPO , TON, and TOF Based on Oxygen
2
CCDC 1992320 and 1992322 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Production from the Decomposition of H2O2 (150 mM) by
Mn(II) Complex of 15-pyN3O2Ph
TOF (min−1
)
ΔPO (mmHg)
TON
15(1)
1 min
0.4(1)
10 min
0.92(4)
20 min
0.61(7)
60 min
0.21(3)
2
67(4)
AUTHOR INFORMATION
Corresponding Authors
■
Kayla N. Green − Department of Chemistry and Biochemistry,
Texas Christian University, Fort Worth, Texas 76129, United
States; orcid.org/0000-0001-8816-7646;
Email: kayla.green@tcu.edu
than at 1 min, which indicates that a plateau has been reached.
The turnover number (TON) was calculated for a reaction
period of 60 min, since no significant increase of the signal was
observed after this point.
́
Gyula Tircso − Department of Physical Chemistry, University of
Table 5 shows the ΔPO for three different trials, turnover
Debrecen, Debrecen, Hungary H-4032; orcid.org/0000-
2
0002-7896-7890; Email: gyula.tircso@science.unideb.hu
number (TON), and turnover frequencies at different times.
Figure 8 shows the O2 evolution signal measured by the O2
Authors
microsensor probe. Both ΔPO and TON were calculated at the
2
Kristof Pota − Department of Chemistry and Biochemistry,
Texas Christian University, Fort Worth, Texas 76129, United
States; orcid.org/0000-0002-9027-0201
end of a reaction period of 60 min, but a constant increase of
the signal was observed, which could be attributed to the
natural disproportionation of H2O2. To determine the rate of
O2 evolution for TOF calculations different slopes were
calculated at several times (1, 10, 20, and 60 min); in the first
minute, several bubbles form in the solution and slowly diffuse
out of it, the rate keeps increasing for a few minutes and then
starts to decrease at 60 min where the plateau is reached. After
that, no more catalytic activity was observed. Overall, the
results indicate that the complex is indeed a functional mimic
of Mn(CAT) but has limited activity under the conditions
studied.
̋
́
Eniko Molnar − Department of Physical Chemistry, University
of Debrecen, Debrecen, Hungary H-4032
́
́
́
Ferenc Krisztian Kalman − Department of Physical Chemistry,
University of Debrecen, Debrecen, Hungary H-4032;
orcid.org/0000-0001-9000-2779
David M. Freire − Department of Chemistry and Biochemistry,
Texas Christian University, Fort Worth, Texas 76129, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01053
CONCLUSIONS
■
Notes
In summary, we have shown that the inclusion of a second
aromatic ring into the backbone of a 15-membered macro-
cyclic system leads to a formation of a surprisingly flat
manganese(II) complex with two water molecules bound in
the solid state. This structural feature plays a key role in
increasing the longitudinal relaxivity (r1p) of the chelate
compared to its parent molecule, due to the open axial
positions around the seven-coordinate metal center. It must be
mentioned, that [Mn(15-pyN3O2Ph)]2+ has a transverse
relaxivity of 11.72 mM−1 s−1 at 0.49 T (298 K), which is
boosted to 25.69 mM−1 s−1 at 1.41 T (298 K), suggesting that
the complex might act as an efficient T2 shortening agent.
These results are supported by a wide array of well-established
experimental techniques (i.e., pH-potentiometric, XRD, 17O
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.P. is grateful for the support from the Fulbright Foundation
and the College of Science and Engineering at TCU. This work
was funded by the National Institutes of Health
R15GM123463 (to K.N.G.). The authors are also grateful
for generous financial support from TCU Andrews Institute of
Mathematics and Science Education (to K.N.G.), Cambridge
Isotope Laboratories, and TCU Research and Creativity
Activity Grant (to K.N.G.). The authors (E.M., F.K.K., and
G.T.) acknowledge financial support from the Hungarian
National Research, Development and Innovation Office
(NKFIH K-120224 project) and the COST Action CA15209
European Network on NMR Relaxometry. The research was
also supported in a part by the EU and cofinanced by the
European Regional Development Fund under the project
GINOP-2.3.2-15-2016-00008.
1
NMR and H relaxometric measurements). Unfortunately, it
has to be noted that as an adverse effect of rigidification, the
Mn(II) chelate is unsuitable for further investigations as a
contrast agent due to its labile nature under even mildly acidic
conditions. However, we have also demonstrated its activity as
an H2O2 disproportionation catalyst, which creates new
opportunities for Mn(CAT) mimics with macrocyclic ligands.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01053.
■
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