Seven coordinate Co(ii) and six coordinate Ni(ii) complexes of an aromatic macrocyclic triamide ligand as paraCEST agents for MRI

Article abstract of DOI:10.1039/c9dt00747d

We are reporting Co(ii) and Ni(ii) complexes of a pyridine containing aromatic macrocyclic triamide ligand, 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetamide (TPTA), as paramagnetic chemical exchange saturation transfer (paraCEST) MRI contrast agents. The synthesis and characterization of TPTA and its complexes are reported. The solution chemistry and solid-state structure of Co(ii) and Ni(ii) complexes are studied. Crystallographic data show that the [Co(TPTA)]·Cl₂·2H₂O complex (seven-coordinate, all four N atoms of ring and three amide O atoms) has a distorted pentagonal bipyramidal geometry, however the [Ni(TPTA)Cl]·Cl·0.25H₂O complex (six-coordinate, all four N atoms of the ring, one amide O and one chloride ion) adopts a distorted octahedral geometry. Notably the two pendent amide arms are not coordinated in the [Ni(TPTA)Cl]⁺ complex and one chloride ion fulfils its sixth coordination. The CEST effect of [Co(TPTA)]²⁺ and [Ni(TPTA)Cl]⁺ amide protons is observed at 57 ppm and 78 ppm downfield of the bulk water proton respectively in a buffer solution containing 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid and 100 mM NaCl at pH 7.4 at 37 °C on a 9.4 T NMR spectrometer. The effects of CEST intensity and exchange rate constant with variation of pH of the solution were studied. The CEST effect and exchange rate constant for the amide protons of the [Co(TPTA)]²⁺ complex have been monitored in HEPES buffer, fetal bovine serum (FBS), rabbit serum and 4% agarose gel (w/w). The stability of the [Co(TPTA)]²⁺ complex in aqueous solution towards oxidation was verified by cyclic voltammetry measurement. The stability of [Co(TPTA)]²⁺ has further been monitored in the presence of biologically relevant ions including HPO₄^2-, CO₃^2-, and Zn²⁺ and under acidic conditions.

Full text of DOI:10.1039/c9dt00747d

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January 2016 Pages 1–398
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DOI: 10.1039/C9DT00747D
Seven Coordinate Co(II) and Six Coordinate Ni(II) Complexes of
Aromatic Macrocyclic Triamide Ligand as ParaCEST Agents for
MRI†
a
b
c
c
Rabindra N. Pradhan, Subhayan Chakraborty, Pratibha Bharti, Janesh Kumar, Arindam
b
a
Ghosh,* and Akhilesh K. Singh*
aSchool of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, 752050
India), E-mail: aksingh@iitbbs.ac.in
(
b
School of Chemical Sciences, National Institute of Science Education and Research
Bhubaneswar, 752050 (India), E-mail: aringh@niser.ac.in
c
National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune-
4
†
11 007 (India)
Electronic supplementary information (ESI) available: 1H NMR, electrospray ionization (ESI)-
MS, magnetic susceptibility data, Cyclic voltammogram, MTT assay, Omega plot, CCDC
884844 and 1884845.
1
We are reporting Co(II) and Ni(II) complexes of a pyridine containing aromatic macrocyclic
triamide ligand, 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetamide
(TPTA) as paramagnetic chemical exchange saturation transfer (paraCEST) MRI contrast agent.
The synthesis and characterization of TPTA and its complexes are reported. The solution
chemistry and solid-state structure of Co(II) and Ni(II) complexes are studied. Crystallographic
.
.
data show that [Co(TPTA)] Cl 2H O complex (seven-coordinate, all four N atoms of ring and
2
2
three amide O atoms) has distorted pentagonal bipyramidal geometry however
.
.
[
Ni(TPTA)Cl] Cl 0.25H O complex (six-coordinate, all four N atoms of the ring, one amide O and
2
one chloride ion) adopts distorted octahedral geometry. Notably the two pendent amide arms are
+
1
not coordinated in [Ni(TPTA)Cl]
complex and one chloride ion fulfils its sixth coordination.
+
2
+1
The CEST effect of [Co(TPTA)] and [Ni(TPTA)Cl] amide protons are observed at 57 ppm
and 78 ppm downfield of the bulk water proton respectively in buffer solution containing 20 mM
N-(2-hydroxyethyl)piperazine-N’-ethanesulfonic acid and 100 mM NaCl at pH 7.4 at 37 ºC on a
9
.4 T NMR spectrometer. The effect of CEST intensity and exchange rate constant with variation
of pH of the solution were studied. The CEST effect and exchange rate constant for amide protons
+
2
of [Co(TPTA)] complex have been monitored in HEPES buffer, fetal bovine serum (FBS), rabbit
+
2
serum and 4% agarose gel (w/w). The stability of the [Co(TPTA)] complex in aqueous solution
towards oxidation was verified by using cyclic voltammetry measurement. The stability of
+
2
[
Co(TPTA)] has further been monitored in presence of biologically relevant ions including
-2
-2
+2
HPO , CO , Zn and under acidic conditions.
4
3
Introduction
Magnetic resonance imaging (MRI) is one of the most significant accepted non-invasive diagnostic
imaging tool for in vivo application. The main advantage of MRI is lack of non-ionizing radiation

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DOI: 10.1039/C9DT00747D
and excellent depth penetration offering high resolution images of soft tissues.1-5 The majority of
the clinically approved contract agents are based on difference in the proton density and T1 and
6
-9
T2 relaxation rates of water protons present in the tissue and organs. So far the conventional
contrast agents are lanthanide based complexes, typically Gd(III) complexes, that shorten the
1
0
longitudinal (T1) relaxation times of water protons and provide high resolution images. The first
generation of contrast agents have limited physical properties and their preinjection and
1
1, 12
postinjection scans are time-consuming, although these are widely used for clinical medicine.
Over the past decades the new generation of MRI contrast agents was devolved which was
operated by chemical exchange saturation transfer (CEST) techniques, able to provide spatial
1
3-16
resolution and metabolic informations.
In order to observe CEST effect the chemical shift
difference between bulk water protons and exchangeable protons (Δω) must be equal or higher
1
1, 12, 17
than the rate of chemical exchange (k ) between two pools.
ex
For last few years, it has been observed that divalent transition metal ion complexes of
macrocyclic and acyclic ligands as paramagnetic chemical exchange saturation transfer
1
8-24
(paraCEST) contrast agents have been of great interest in magnetic resonance imaging (MRI).
This type of contrast agents provide potential advantages over lanthanide, Ln(III) contrast agents.
The discrete coordination chemistry of first row transition metal ions motivates to develop new
type of paraCEST contrast agents which provide opportunities for application in broad area of
MRI imaging. Co(II), Fe(II), and Ni(II) complexes are mostly used as effective paraCEST contrast
1
8-20, 24
agents among all transition metal ion complexes.
The paramagnetic centre interacts with
ligand protons not only through bond but also through space to produce sharp and highly shifted
1
25-27
H proton resonance on the NMR time scale.
The paramagnetic metal complexes that show
paraCEST properties must be associated with exchangeable protons (-NH, -OH, or bound water)
2
8-33
that exchange with the bulk water.
To get contrast in presence of paraCEST agents, selective
irradiation of radiofrequency pulse is applied to a pool of exchangeable protons which are in slow
to intermediate exchange with bulk water protons which causes decrease in the magnetization of
these exchanging protons. This phenomena leads to decrease in the water proton signal.11, 17, 34, 35
In order to produce thermodynamically stable and kinetically inert metal-based paraCEST probe
at physiological pH and temperature, typically the metal complexes of ligands containing N and
2
2-24, 36
20, 21
O donors are used. The multidentate ligands form rigid six-
, seven-
and eight-
1
9, 21
coordinate
broadening and large proton shift in H spectra.
complexes with Fe(II), Co(II), or Ni(II) making it possible to produce limited
1
25, 26
The ligands must contain hetero atom donor
groups like amides, alcohol, pyridines, imidazole and pyrazoles with exchangeable protons that
chemically exchange with water. Morrow and other research groups have reported several
2
1, 24
macrocyclic ligands with different pendent arms (1,4,7-triazacyclononane (tacn)
, 1,4,7,10-
1,4,8,11-tetraazacyclotetradecane (cyclam), 1,4,10-trioxa-
)) derivatives, whereas Harris and co-workers have reported
1
9, 37
22
tetraazacyclododecane (cyclen),
1
8, 20
7
,13-diazacyclopentadecane
2
3, 38
amide-appended acyclic ligands (2-hydroxy-5-nitro-1,3-phenylene)bis(methylene)
transition metal complexes provide stable and efficient paraCEST contrast agents.
whose
In the present work, we introduce for the first time a novel pyridine containing macrocyclic
backbone 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetamide ligand
(TPTA, Chart 1) for transition metal paraCEST contrast agent with amide donor groups that
produce a CEST effect through exchange. Herein, we report the synthesis and characterisation of

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.
.
the ligand and its corresponding metal complexes [Co(TPTA)] Cl 2H O
[
and
2
2
.
.
Ni(TPTA)Cl] Cl 0.25H O. Effective magnetic moment in solution state and CEST properties of
2
the both structurally characterized complexes were investigated.
The dissociation of
+
2
[Co(TPTA)] complex is studied in acidic condition as well as in presence of biologically relevant
+
2
ions such as carbonate, Zn(II) and phosphate. The redox behaviour of [Co(TPTA)] complex was
+
2
investigated in aqueous solution. The exchangeable carboxamide protons of [Co(TPTA)]
complex show noticeable change in CEST properties with variation of pH. The maximum CEST
+
2
+2
intensity of the [Co(TPTA)] complex is observed at pH 7.8. Interestingly the [Co(TPTA)]
complex retains its CEST activity in different media including rabbit serum, fetal bovine serum
FBS) and 4% agarose gel (w/w), which makes it suitable for potential application in living
(
systems. The biocompatibility has been evaluated using tissue culture in HEK cell, which shows
no toxicity up to 10 mM concentration.
NH2
N
O
N
N
O
H N
N
2
H N
2
O
TPTA
Chart 1 Structure of ligand TPTA
Results and discussion
Synthesis and crystal structure
The macrocylic 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (3) was prepared by
3
9
slight modification of the previously reported procedure which involved the synthesis of 3,6,9-
tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene (2) and
subsequent deprotection of the tosyl groups by sulfuric acid. Then alkylation of the three secondary
amine groups of 3 with 2-bromoacetamide yielded the ligand TPTA. The corresponding metal
complexes were isolated by addition of stoichiometric amounts (1:1) of the ligand and the chloride
salts of Co(II), and Ni(II) in methanol (see Experimental Section for details). To explore the
coordination environment around central metal ions of these complexes, their structures were
characterized by single crystal X-ray diffraction.
.
.
Slow evaporation of diethyl ether vapour into concentrated solution of [Co(TPTA)] Cl 2H O
2
2
in MeOH afforded pink coloured block like crystals and slow evaporation of
.
.
[
Ni(TPTA)Cl] Cl 0.5H O in a mixture of DMF and MeOH yielded plate like bluish crystals. X-
2 2 2
+
2
+2
ray diffraction data indicated that both complexes [Co(TPTA)] and [Ni(TPTA)Cl] adopted
triclinic unit cell with a space group of P-1 (Table 2 in Experimental section). In [Co(TPTA)]
+
2
all nitrogen atoms (N1, N2, N4, N6) of macrocyclic backbone and oxygen atoms (O1, O2, O3) of
the pendent carboxamide groups formed a seven coordinate complex. The coordination polyhedra

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around the metal ion (Co(II)) can be best described as a distorted pentagonal bipyramidal geometry
Fig. 1). Three amine-N atoms (N2, N4 and N6) of macrocyclic ring and two amide-O (O1 and
(
O3) of the pendent arms form the pentagonal base, and the pyridine-N atom (N1) of the
macrocyclic backbone and the amide-O (O2) of remaining pendent arm occupy the axial positions
+
2
(
Fig. 1). [Co(TPTA)] complex has two types (axial and equatorial) of metal-nitrogen and metal-
oxygen bonds. The axial Cobalt-nitrogen (pyridine) (Co1-N1) bond is shorter than other Cobalt-
nitrogen bonds in the macrocyclic backbone. The average Co1-O and Co1-N bond distances of
+
2
[
Co(TPTA)] are 2.185(5) Å and 2.227(5) Å, respectively. The axial bond angle of N1-Co1-O2
is 172.9(2), confirms that both N1 and O2 are in axial position of the pentagonal bipyramidal
structure (Fig. 1 and Table S2, ESI†). The overall charge balance for Co(II) is confirmed by the
presence of two chloride anions.
.
.
Fig. 1 X-ray crystal structural of the [Co(TPTA)] Cl 2H O complex. For clarity, the solvent, hydrogen and
2
2
counter ions were omitted in the structure.
.
.
The [Ni(TPTA)Cl] Cl 0.5H O complex has six coordinated metal centre. The Ni(II) ion is
2
2
2
bound to four nitrogen atoms of macrocyclic ring (N1, N2, N4, N6), one amide oxygen (O1) of
pendent arm and one chloride ion to give a distorted octahedral geometry (Fig. 2). The remaining
two pendent arms of the ligand are dangling and not coordinated to the metal centre. The amide-
O (O1) and the pyridine-N (N1) are oriented in trans position to each other to give a distorted
octahedral geometry. This distortion can be attributed to Jahn-Teller effect. The axial bonds with
bond length of 2.015(3) and 2.083(3) Å for Ni1-N1 and Ni1-O1 respectively are shorter than other
bonds (see in the SI Table S2). The average bond length of other three nitrogen atoms (N2, N4,
N6) is 2.191 (3) Å (see in the SI Table S2) and the bond length of Ni1-Cl1 is 2.336 (3), which is
little longer than other bonds. The important bond lengths and bond angles are given in the
Supporting Information (Table S2).

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.
.
Fig. 2 X-ray crystal structural of the [Ni(TPTA)] Cl 0.5H O complex. For clarity, the solvent, hydrogen and
2
2
2
counter ions were omitted in the structure.
Solution magnetic properties
In order to confirm the dissociation of chloride ion in [Ni(TPTA)Cl]Cl complex, the complex
1:1 electrolyte in solid state) was dissolved in water and its molar conductance was measured.
Most likely the bound chloride ion is dissociated and it is exchanged with water because the
(
-1
2
-1
obtained molar conductance value (192 ohm cm mol ) lie in the range of 1:2 electrolyte (145-
-
1
2
-1 40
2
[
73 ohm cm mol ). In aqueous buffer solutions the effective magnetic moment of
+
2
+1
Co(TPTA)] and [Ni(TPTA)Cl] complexes with variation of pH 6.5-8.4 were obtained at 37
ºC using Evans method (see experimental section). The resulting plot of 휒 푇 versus pH (see Fig.
푀
S23 and S24, ESI†) ensure that the variation of 휒 푇 values is insignificant with variation of pH.
푀
+
2
+1
The average effective magnetic moments of [Co(TPTA)] and [Ni(TPTA)Cl] are obtained 4.07
0.10 and 3.05 ± 0.02 µ respectively. These values are in the expected range for structurally
±
B
reported similar high spin Co(II) and Ni(II) complexes at physiological pH at 37 °C in aqueous
2
6, 27
+2
solution.
The effective magnetic moment of [Co(TPTA)] complex was measured in
biological media like rabbit serum and FBS at pH 7.4 were found to be 4.16 and 4.22 µB
respectively.
NMR spectroscopy
+
2
+1
In order to investigate the solution properties of [Co(TPTA)] and [Ni(TPTA)Cl] complexes,
1
+2
H NMR spectra were collected in DMSO, D O and buffer solutions. In D O [Co(TPTA)]
2
2
,
exhibits total 9 proton resonances ranging from 0 to 170 ppm at 25 °C (Fig. 3), with two
exchangeable carboxamide protons at 63 and 68 ppm, identified by the decrease in intensity of
these peaks observed in the analogous spectrum after addition of D O in DMSO-d (Fig. S16,
2
6
ESI†). Out of these 9 proton resonances, one proton resonance is found within diamagnetic regions
Fig. 3). The 8 paramagnetic resonances have equivalent integrated intensities, while the resonance
(
1
1
within diamagnetic regions has half integrated intensity. The H- H-COSY spectrum allowed us
to identify the resonances at 10.31 and 42.05 in D O as the aromatic protons of the [Co(TPTA)]
+
2
2
(
Fig. S19, ESI†), while the other 7 paramagnetic proton resonances correspond to CH peaks of
2
1
the complex. In the H NMR spectra recorded at variable temperatures ranging from 25 °C to 60

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°
C, the peak widths of the resonances changed very little upon increase in temperature (Fig. S20,
ESI†). This suggests that the dynamic processes do not have much effect on the line broadening
1
over this temperature range. The H NMR resonances of two paramagnetic CH peaks at 167.5
2
and 107.8 in D O are highly-shifted towards upfield with increase in the temperature from 25 °C
2
4
0
1
to 60 °C (Fig. S21, ESI†), similar to the reported Co(II) complex. However, H NMR spectra
shows significant broadening of the exchangeable protons when pH of the solution is increased in
the range of 6.10 to 8.40 for the [Co(TPTA)] complex, confirming base catalysed proton
+
2
2
3, 37
exchange
(Fig. S25, ESI†).
1
+2
Fig. 3 H NMR spectrum of [Co(TPTA)] in deuterium oxide.
+
1
In comparison, the proton NMR spectrum of [Ni(TPTA)Cl] exhibits 7 paramagnetic proton
resonances ranging from 0 to 150 ppm in D O (see Fig. S18 ESI†), with exchangeable
2
carboxamide signals at 7, 13 and 78 ppm, confirmed by comparison of the spectra recorded in
DMSO-d and after addition of D O in DMSO-d (see Fig. S17, ESI†). The resonances of two
6
2
6
unbounded exchangeable carboxamide protons are slightly shifted downfield from the diamagnetic
region at 7 and 13 ppm which were similar to the reported six coordinated Ni(II) complex.22
Additionally one highly shifted amide proton resonance at 78 ppm was observed. Like many Ni(II)
+
1
complexes, the [Ni(TPTA)Cl] complex shows broad peaks due to its long electronic relaxation
4
2
time.
Redox properties
+
2
To investigate the inertness of the [Co(TPTA)] complex in solutions towards oxidation, cyclic
voltammetry experiment was performed in an aqueous buffer solution at pH 7.4. The
+
2
[
Co(TPTA)] complex shows an irreversible oxidative response at E(peak) = 792 mV versus
NHE which corresponds to Co(III)/Co(II) redox couple (see Fig. S39, ESI). The higher positive

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+
2
value for the oxidative response suggests the stability of [Co(TPTA)] complex towards
4
3
oxidation in aqueous solution.
Dissociation of complex
+
2
1
To determine the dissociation of the [Co(TPTA)] complex, H NMR spectra of the complex in
paramagnetic and diamagnetic regions were monitored. Under acidic condition (pD-3.96) the
+
2
[
Co(TPTA)] complex dissociated by 6% over 72 h at 37 °C (see Fig. S27 and S28, ESI†). In
presence of equimolar concentration of competing Zn(II) ions at 37 °C, pD-7.01 for 72 h ,
+
2
[
Co(TPTA)] complex did not show any evidence of metal ion dissociation, as the intensity of
proton resonances remain constant as compared to the standard at neutral pH (see Fig. S27 and
+
2
S30, ESI†). In order to further determine the stability of [Co(TPTA)] complex in the presence
of high concentrations of carbonate and phosphate under physiological condition, 10 mM complex
at pD 7.34 was incubated with 100 mM NaCl, 25 mM K CO , 0.40 mM Na HPO at 37 °C and
2
3
2
4
1
was monitored for 72 h. The H spectra were recorded at different time interval revealed that the
complex does not dissociate even in the presence of high concentration of anions (see Fig. S27 and
S29, ESI†). Notably, the intensity of proton resonances decreased under these conditions which
indicates some interaction with the anions, but such interaction did not alter the CEST efficiency
+
2
(
see Fig. S37, ESI†). Taken altogether, the stability of [Co(TPTA)] complex is very high under
physiological conditions which paves the way of its potential application as an MRI contrast agent
+
1
for in vivo studies. As the two pendent amide arms in [Ni(TPTA)Cl] are not coordinated to the
+
2
+2
Ni and this complex exhibits very low CEST intensity as compared to [Co(TPTA)] , the
[
+
1
+2
Ni(TPTA)Cl] complex could not be studied in a similar way like the [Co(TPTA)] complex.
Biological characterizations
+
2
To investigate the cytotoxicity in vitro the [Co(TPTA)] complex was examined using MTT assay
on HEK 293 WT cell line. The cells were incubated at different concentration of the complex (1-
1
0 mM) for 24 h at 37 °C. The complex did not show significant toxicity up to the tested
concentration of 10 mM (see Fig. S38, ESI†). After 24 hours of treatment with the compound, the
cell viability remained unchanged.
CEST properties
+
2
+1
In order to investigate the paraCEST properties of [Co(TPTA)] and [Ni(TPTA)Cl] complexes,
the CEST spectra were collected with 10 mM complexes, 100 mM NaCl, and 20 mM of HEPES
+
2
+1
buffer at 37 °C. The [Co(TPTA)] and [Ni(TPTA)Cl] complexes produced downfield-shifted
peak at 57 and 70 ppm respectively verses bulk water (Fig. 4). CEST peak at 57 ppm downfield
+
2
from bulk water protons for [Co(TPTA)] complex is attributed to the distinct exchangeable
amide (NH) protons at 68 and 63 ppm in DMSO-d ( see Fig. S16, ESI†) and the CEST peak at 70
6
ppm is attributed to the exchangeable amide proton at 78 ppm in DMSO-d (see Fig. S17 ESI†).
6
+
1
The CEST signals for the exchangeable NH protons at 7 ppm for [Ni(TPTA)Cl] complex is
concealed by direct saturation of bulk water protons.

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+
2
+1
Unlike [Co(TPTA)] in buffer medium, the [Ni(TPTA)Cl] complex does not show appreciable
CEST effect due to the fact that the exchange constant and T relaxivity of the complex is larger
1
+
2
than the [Co(TPTA)] complex.
The CEST effect as expected was found to be very sensitive to the pH level of the solution and
+
2
the [Co(TPTA)] complex showed maximum CEST effect at pH 7.8. Fig. 5 compares the z-
spectra (top, the exchangeable proton region around 57 ppm and bottom, CEST effect) at pH
ranging from 6.8 to 8.3. The CEST intensity increases with increase in the pH in the range of 6.8
to 7.8, consistent with the base catalysed proton exchange of carboxamide protons. The gradual
decrease of CEST efficiency at more basic pH is attributed to faster exchange, leading to exchange
broadening for amide protons. Similar pH dependence trends were observed for paraCEST contrast
1
2, 20, 23, 35
agents containing amide pendent for both Ln(III) and transition metal ions.
The CEST
peak intensity increases almost linearly with pH before it drops marginally beyond pH 7.8. At
physiological condition (pH 7.4 and 37 °C) the CEST effect was found to be 30% (Fig. 4). CEST
effects were calculated by measuring the difference in normalized water peak intensities from a
pair of experiments, one with on resonance saturation of the exchangeable proton (at 57 ppm) and
the other by off-resonance saturation at the symmetrically opposite frequency of –57 ppm with
respect to bulk water.
Fig. 4 Overlaid CEST z-spectra of 10 mM complexes [Co(TPTA)]+2 (blue) and [Ni(TPTA)Cl]+1 (red)) in 20 mM

HEPES buffer and 100 mM NaCl at pH 7.4. RF presaturation pulse was applied for 2 s with B
1
= 25µT at 37 C.
+
2
The exchange rate constant k of amide (NH) protons of 10 mM [Co(TPTA)] and 40 mM
[
ex
+
1
Ni(TPTA)Cl] complexes at 37 ºC in buffer medium containing 20 mM HEPES and 100 mM

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-1
-1
NaCl at pH 7.4 were found to be 3691 s and 5096 s respectively (Fig. S32 and S34, Table S1,
ESI†). The exchange rate constant of the Ni(II) complex is nearly three times larger than that of
Co(II) complex, so the CEST effect of the Ni(II) complex would be anticipated to decrease. The
+
2
exchange rate constant of [Co(TPTA)] increases with increase in the pH in the range of 6.8 to
.3, consistent with the base catalysed proton exchange of carboxamide protons (Fig. S31, S32
and S33, Table S1, ESI†). At higher pH value of 8.3, rapid proton exchange leads to exchange
8
1
2, 19, 22-24, 36, 44-46
broadening and a decrease of the CEST peak.
6
The exchange rate constant at pH
-1
-1
.8 was found 2379 s which is very less as compared to the exchange rate constant 13380 s at
pH 8.3 (see Fig. S31 and S33, Table S1, ESI†).
Fig. 5 Dependence of CEST effect on pH. (Top) Overlaid spectra of the exchangeable proton region of 10 mM
+
2
[
Co(TPTA)] in 20 mM HEPES buffer with pH ranging from 6.8 to 8.3. (Bottom) CEST peak intensity as a function
of pH as calculated from the spectra shown in top. All experiments were performed with 2 s presaturation
radiofrequency pulse at B = 25 µT.
1

Dalton Transactions
Page 10 of 22
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DOI: 10.1039/C9DT00747D
+
2
To monitor the efficiency of the [Co(TPTA)] complex as a CEST agent in biological media,
+
2
separate 10 mM solutions of the [Co(TPTA)] complex were prepared in rabbit serum, fetal
bovine serum (FBS) and 4% agarose gel at pH 7.4. All spectra were recorded at 37 °C. Fig. 6
+
2
shows a comparison of z-spectra of the [Co(TPTA)] complex in HEPES buffer as well as in the
three biological media. The exchange rate constant (k ) were measured in all three biological
ex
media (SI Fig. S35, ESI†) and compared those with aqueous buffer solution (Fig. S32, ESI†). The
CEST effect in the physiological condition were found marginally lower in rabbit serum (28%),
FBS(25%) and agarose gel (23%) in comparison to the buffered medium (30%). The decrease in
CEST effect in rabbit serum by (2%), FBS by (5%), and agarose gel by (7%) with respect to
buffered medium can be attributed to the decrease of the exchange rate constant of the amide
-
1
-1
protons. The k in rabbit serum, FBS and agarose gel were found to be 3286 s , 3643 s , and 3203
ex
-1
s respectively. The T relaxivity studies showed significant change in longitudinal relaxation rate
constant to water protons in presence of the [Co(TPTA)] complex. The measured relaxivities
were 0.66 mM s in 20 mM HEPES buffer, 0.28 mM s in rabbit serum, 0.05 mM s in FBS
and 0.06 mM s in 4% agarose gel (Fig. S40-S47, ESI†). The T1 relaxivity of [Ni(TPTA)Cl]
complex was found 0.90 mM s in 20 mM HEPES and 100 mM NaCl. The differences in
relaxivity of [Co(TPTA)] complex do not contribute significantly to the observed differences in
CEST peak intensity for different media. It is evident that the CEST intensity of the [Co(TPTA)]
1
+
2
-1
-1
-1 -1
-1 -1
-1
-1
+1
-1
-1
+
2
+
2
complex in different media mostly depends upon the exchange rate constant of the carboxamide
protons. In case of agarose gel the CEST spectra is quite different from other media, as it shows
large MT effect between –90 to +90 ppm (Fig. 5). The percentage of CEST effect was calculated
by measuring the difference in normalized water peak signal (Mz/M %) at +57 ppm and -57 ppm
0
with respect to bulk water.
The CEST effect at pH 7.4 and at 37 ºC was found to be gradually diminishing with decreasing
concentration of the complex from 30% at 10 mM to 17% at 3 mM (see Fig. S36, ESI†). However,
the complex did not show any appreciable change in CEST effect (negligible dissociation) within
+
2
the first 24 hours of preparing a solution containing 10 mM [Co(TPTA)] , 100 mM NaCl, 0.4
mM Na HPO , 25 mM K CO at pH 7.80 (Fig. S37, ESI†).
2
4
2
3

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Fig. 6 Overlaid CEST z-spectra of solutions containing 10 mM [Co(TPTA)]+2 in 20 mM HEPES buffer and 100 mM
NaCl at pH 7.4 (magenta), in rabbit serum at pH 7.4 (blue), in FBS at pH 7.4 (green) and in 4% agarose gel, 20 mM
HEPES buffer and 100 mM NaCl at pH 7.4 (red).
Table 1. Comparison of Co(II) and Ni(II) complexes of TPTA and similar analogues.
Δωa
CEST %
k (S )
-1
T relaxivity
1
complex
ex
-1 -1
(
Mm S )
[
[
[
Co(TPTA)]+2
Co(CYNO)]2+
Co(TCMC)]2+
57
59
45
112
70
30b
38c
21.0 ± 0.1d
31.5 ± 0.2e
3691f
0.66 j
240 ± 70g
300h
0.038k
0.096l
0.008l
Co(CCRM)]2+
Ni(TPTA)Cl]+1
Ni(CYNO)]2+
Ni(TCMC)]2+
NI(CCRM)]2+
510i
5096f
[
[
[
[
[
5b
.90j
0
72
76
76
39 ± 0.2c
12.7 ± 0.9d
14e
240 ± 20g
0.012k
328i
0.097l
a
The chemical shift of the furthest downfield shifted amide (NH) exchangeable proton versus the water proton
b
resonances. 10 mM complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, B
radiofrequency pulse at 37 °C, measure on 9.4 T NMR, 10 mM complex, 100 mM NaCl, and 20 mM of HEPES
buffer pH 7.4, B = 24 µT, 2 s presaturation radiofrequency pulse at 37 °C, measure on 11.7 T NMR. 10 mM
1
1
= 25 µT, 2 s presaturation
c
d,e
complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, B
1
= 24 µT, 2 s presaturation radiofrequency pulse at
f
3
9
7 °C, measure on 11.7 T NMR. 10 mM complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, at 37 °C, on
.4 T NMR. 8 mM complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, at 37 °C, on 11.7 T NMR. 10
g
h, i
j
mM complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, at 37 °C, measure on 11.7 T NMR 10 mM
complex, 100 mM NaCl, and 20 mM of HEPES buffer pH 7.4, at 37 °C, on 9.4 T NMR. 8 mM complex, 100 mM
NaCl, and 20 mM of HEPES buffer pH 7.4, at 37 °C, on 11.7 T NMR. 10 mM complex, 100 mM NaCl, and 20 mM
k
l
of HEPES buffer pH 7.4, at 37 °C, on 4.7 T NMR.
It is of interest to compare the CEST spectra of Co(II) and Ni(II) complex of TPTA ligand with
similar Co(II) and NI (II) macrocyclic ligands in the literature (Table 1). The comparison is made
with the furthest shifted CEST peak of Co(II) and Ni (II) complexes of previously reported ligands

Dalton Transactions
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DOI: 10.1039/C9DT00747D
2
0
7
,13-bis(carbamoylmethyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (CYNO) , 1,4,7,10-
1
2, 21
tetrakis (carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC)
tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (CCRM).
and 1,4,8,11-
1
2, 21
Conclusions
We have designed and synthesized a new pyridine containing heptadentate macrocyclic triamide
+
2
+1
ligand (TPTA) for studying the paraCEST properties. [Co(TPTA)] and [Ni(TPTA)Cl]
complexes have been prepared and their properties have been studied in solution and solid-state.
+
2
1
On NMR time scale [Co(TPTA)] complex produces sharp and highly shifted H resonances.
Coordination features reveal that Co(II) is seven coordinated with distorted pentagonal
bipyramidal geometry and Ni(II) is six coordinated with distorted octahedral geometry. In
+
2
[
Co(TPTA)] complex all nitrogens of the ring and three amide oxygens from the pendent arms
are coordinating to the metal centre whereas in Ni(II) complex two pendent amide arms are not
+
2
coordinating to the metal centre. The [Co(TPTA)] complex has shown sharp and intense CEST
+
2
contrast in buffer, rabbit serum, FBS and agarose gel. The CEST experiment of [Co(TPTA)]
+
2
complex in agarose gel shows large MT effect. The [Co(TPTA)] complex is highly stable in
presence of biologically competing anions (high concentration of carbonate and phosphate) and
+
2
competing metal ion ( Zn(II)). The cyclic voltammetry experiment of [Co(TPTA)] proves that
the complex is inert toward oxidation in solution. Most interestingly, the cell viability experiment
+
2
of [Co(TPTA)] complex shows that the complex is almost nontoxic up to 10 mM concentration.
+
2
Overall the [Co(TPTA)] complex is highly stable in all conditions, nontoxic and gives intense
CEST effect in all biological media which suggests that the complex may be useful for in vivo
applications.
Experimental Section
Materials and methods
.
All the reactions were carried out at ambient atmosphere and temperature. CoCl 6H O,
2
2
.
NiCl 6H O, rabbit serum, agarose gel were purchased from Merck. Deuterated solvents were
2
2
purchased from Sigma-Aldrich. All other chemicals and reagents (analytical and spectroscopic
grade) were obtained from commercial sources and used as received. Acetonitrile (MeCN),
methanol (MeOH), N,N-Dimethylformamide (DMF), ethanol (EtOH) and diethyl ether (Et O)
2
were dried using standard literature methods. pH dependant magnetic measurements of complexes,
NMR experiments and dissociation of complexes were carried out by using Expert pro-ISM-IP67
®
electrode connected to METTLER TOLEDO InLab 73x series pH meter. pH of CEST experiment
was maintained by using Thermo Scientific electrode connected to EUTECH Instruments pH
Tutor pH/°C meter. BUCHI M-560 instrument was used to measure the melting points. Elemental
analysis was performed using an Elementar vario MICRO cube CHN analyzer. Electro chemical
measurements were performed using a CHI 620D electrochemical analyzer equipped with a glassy
carbon working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference
electrode. All the infrared (FT-IR) data were collected with KBr pellets on a BRUKER ALPHA-
T FT-IR spectrometer. Perkin-Elmer model Lambda 650 UV-Vis spectrophotometer was used to

Page 13 of 22
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DOI: 10.1039/C9DT00747D
record all absorption spectra of ligands and complexes. Evans’ measurement, dissociation of
1
13
complex at different conditions and H and C NMR spectra were acquired on Bruker AVANCE
00 NMR spectrometer. Bruker microTOF-Q II mass spectrometer was used to collect the mass
4
(ESI-MS) spectra of ligand and complexes.
Ts
N
TEA
KI
dry EtOH
NH2
N
N
HN
TS TS
NH
N
Conc.
H SO
2 4 HN
O
N
N
N
Ts
N
Ts
N
N
NH
O
Br
Br
O
dry DMF,
K CO
2
H N
2
N
Ts
H N
2
3
N
H
NH2
Br
O
TPTA
1
2
3
Scheme 1 Synthetic scheme of ligand TPTA.
Synthesis of Compound (2). The compound 1 was synthesized by esterification of 2-6-dipicolinic
acid and then reduction with sodium borohydride followed by bromination with PBr using the
3
4
7
reported literature method. The compound 2 was synthesized by following the reported literature
method with slight modification. A mixture of N, N, N"-Tri(toluene-4-sulfonyl)diethylene
3
9
triamine (3.0 g, 5.30 mmol) and K CO (2.2 g, 15.9 mmol) were mixed together in dry DMF (100
2
3
mL) and the mixture was stirred and heated to 100 °C under N atmosphere. A solution of 2,6-
2
bis(bromomethyl)pyridine (1) (1.4 g, 5.30 mmol) in dry DMF (30 mL) was added dropwise over
3
0 min. When the addition was completed, the reaction mixture was stirred at 50 °C under N2
atmosphere for 24 h. After cooling, the reaction mixture was poured into water (75 mL). The
resulting precipitate was then filtered and the solid was washed with water and dried in vacuum.
The crude product was purified by column chromatography (SiO CHCl /EtOAc = 30:1) to give
2
,
3
the desired product 2 (2.84 g, 79%) as white powder. mp: 172-174.5 °C. Anal. Calcd for
C H N O S : C, 57.47; H, 5.43; N, 8.38; S, 14.38%. Found: C, 57.83; H, 5.79; N, 7.88; S;
3
2
36
4
6 3
+
1
4.17%. ESI-MS m/z: calcd for C H N O S ([MH] ), 669.18; found, 669.17; calcd for
C H N NaO S ([MNa] ), 691.17; found: 691.17. H NMR (400 MHz, CDCl , 25 °C): δ 2.42 (s,
3
32 37 4 6 3
+
1
2
36
4
6
3
3
3
H), 2.47 (s, 6H), 2.78 (s, 4H), 3.35 (t, J = 7.2 Hz 4H), 4.31 (s, 4H), 7.29 (d, J = 8.4 Hz, 2H), 7.36
(
d, J = 8.4 Hz, 4H), 7.45 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 8.4 Hz, 4H),
.78 (d, J = 8.4 Hz, 1H). C NMR (100 MHz, CDCl 25 °C): δ 155.3 (Ar-CH ), 143.8 (Ar-SO ),
3, 2 2
1
3
7
1
1
43.5 (Ar-SO ), 139.0 (Ar-H), 136.0 (Ar-CH ), 135.2 (Ar-CH ), 130.0 (Ar-H), 129.8 (Ar-H),
2
3
3
27.2 (Ar-H), 127.1 (Ar-H), 124.3 (Ar-H), 55.0 (Ar-CH -N), 47.4 (N-CH -CH -N), 21.6 (Ar-
2
2
2
-1
CH ), 21.5 (Ar-CH ). FT-IR (KBr, cm ): 3439 (w, broad); 2925 (w); 1594 (m); 1455 (s); 1358 (s);
3
3
1
156 (s); 1094 (s); 949 (m); 815 (m); 760 (m); 682 (m); 650 (m); 551 (s).
Synthesis of Compound (3). A mixture of 2 (1 g, 1.29 mmol) and conc. sulfuric acid (10 mL)
was stirred at 115-120 °C for 2 h. The reaction mixture was transferred into an erlenmeyer flask
having cracked ice, then neutralized by slow addition of 40% NaOH solution until pH reached to
8
. The product was extracted with CHCl (50 mL) in three portions. The organic phase was dried
3
over MgSO and was evaporated to give 3 (0.310g, 80%) as a pale yellow solid. ESI-MS m/z:
4
+
+
calcd for C H N ([MH] ), 207.15; found, 207.16; calcd for C H NaN ([MNa] ), 229.14;
1
1
19
1
4
11 18
4
found: 229.14. H NMR (400 MHz, CDCl , 25 °C): δ 2.23 (s, 4H), 2.67 (t, J = 4.8 Hz, 2H), 2.93
s, 3H), 3.94 (s, 4H), 6.98 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 8.4 Hz, 1H). C NMR (100 MHz, CDCl
3
1
3
(
3,

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DOI: 10.1039/C9DT00747D
2
4
5 °C): δ 159.8 (Ar-CH ), 136.4 (Ar-H), 119.9 (Ar-H), 53.9 (Ar-CH -N), 49.3 (N-CH -CH -N),
2 2 2 2
-1
9.0 (N-CH -CH -N). FT-IR (KBr, cm ): 3298 (s); 2883 (m); 1577 (s); 1461 (s); 1400 (m); 1345
2
2
(
m); 1300 (m); 1124 (m); 790 (m).
Synthesis of Ligand (TPTA). The macrocyclic compound 3 (0.240 g, 0.84 mmol), KI (0.153 g,
.92 mmol) and 2-bromoacetamide (0.463 g, 3.36 mmol) were mixed together in dry ethanol (15
0
mL) followed by addition of trimethylamine (1 mL, 7.56 mmol). The mixture was heated to 85 °C
for 72 h under N atmosphere. Upon completion of reaction, solvent was removed under reduced
2
pressure producing a brown oily residue. The residue was purified by column chromatography on
neutral alumina eluting with dichloromethane/methanol (20:1 ratio) to give a light yellow liquid.
The light yellow liquid was further purified by dissolving the product in methanol (5 mL) and
dropwise addition of diethyl ether to precipitate the product. The precipitate was isolated by
filtration, washed with diethyl ether and dried in vacuo to give TPTA (0.198g, 52%) as a white
powder. mp: 237-239 °C. Anal. Calcd for C H N O : C, 54.10; H, 7.21; N, 25.98%. Found: C,
1
7
27
7
3
+
5
4.83; H, 8.03; N, 26.13%. ESI-MS m/z: calcd for C H N O ([MH] ), 378.22; found, 378.22;
calcd for C H N NaO ([MNa] ), 400.21; found: 400.21. H NMR (400 MHz, D O, ppm): δ =
17 28 7 3
+
1
1
7
27
7
3
2
2
.01 (s, 4H), 2.51 (s, 4H), 2.97 (s, 2H), 3.31 (s, 4H), 3.70 (s, 4H), 7.18 (d, J = 7.6 Hz, 2H), 7.68 (t,
1
3
J = 7.6 Hz, 1H), C NMR (100 MHz, D O, ppm): 176.2 (C=O), 168.8 (C=O), 158.3 (Ar-CH ),
2
2
1
38.9 (Ar-H), 121.1 (Ar-H), 60.7 (Ar-CH -N), 59.1 (N-CH -C=O), 56.0 (N-CH -CH -N), 52.1 (N-
2 2 2 2
CH -C=O), 51.5 (N-CH -CH -N). UV-vis absorption spectrum (250 μM; 20 mM HEPES, 100 mM
2
2
2
-1
-1
-1
NaCl at pH 7.4, 25 °C): 267 nm (ɛ = 3620 M cm ). FT-IR (KBr, cm ): 3387 (s); 3299 (s); 3182
s); 2925 (m); 2843 (m); 1670 (s); 1617 (s); 1384 (s); 1293 (m); 1234 (w); 1163 (w); 1140 (m);
074 (w); 639 (m).
Synthesis of [Co(TPTA)] Cl 2H O. A solution of CoCl 6H O (63 mg, 0.26 mmol) in dry MeOH
(
1
.
.
.
2
2
2
2
(5 ml) was added dropwise to a stirring white suspension of TPTA (100 mg, 0.26 mmol) in dry
MeOH (10 ml) to give a pink colour solution. The reaction mixture was heated gently and was
stirred at room temperature for 3 h to get a clear solution. The presence of free Co(II) ion was
checked by xylenol orange test. After stirring for 3 h, the solvent was removed and the pink solid
was collected and washed with Et O and dried under vacuum for 12 h to give pink coloured solid
2
.
.
(
107mg, 0.21 mmol) of [Co(TPTA)] Cl 2H O. Anal. Calcd for C H Cl CoN O : C, 37.58; H,
2 2 17 31 2 7 5
5
.75; N, 18.06%. Found: C, 37.51; H, 5.72; N, 17.98%. ESI-MS m/z: calcd for C H N O Co
17 27 7 3
2
+
([CoL] ), 218.07; found, 218.07. UV-vis absorption spectrum (250 μM; 20 mM HEPES, 100 mM
-1
-1
-1
NaCl at pH 7.4, 25 °C): 267 nm (ɛ = 2350 M cm ). FT-IR (KBr, cm ): 3380 (s); 3292 (s); 3164
m); 2925 (m); 2908 (m); 1658 (s); 1610 (m); 1453 (m); 1424 (s); 1384 (m); 1234 (w); 1095 (m);
67 (w); 625 (w).
Synthesis of [Ni(TPTA)Cl] Cl 0.25H O. A solution of NiCl 6H O (63 mg, 0.26 mmol) in dry
(
6
.
.
.
2
2
2
MeOH (5 ml) was added dropwise to a stirring white suspension of TPTA (100 mg, 0.26 mmol)
in dry MeOH (10 ml) to give a bluish colour solution. The reaction mixture was heated gently and
was stirred at room temperature for 3 h to get a clear solution. The presence of free Ni(II) ion was
checked by xylenol orange test. After stirring for 3 h, the solvent was removed and the bluish solid
was collected and washed with Et O and dried in vacuo for 12 h to give bluish solid (107mg, 0.21
2
.
.
mmol) of [Ni(TPTA)Cl] Cl 0.25H O. Anal. Calcd for C H27.5Cl N NiO : C, 37.53; H, 5.41;
2
17
2
7
3.25
2
+
N, 19.16%. Found: C, 36.84; H, 5.39; N, 18.58%. ESI-MS m/z: calcd for C H N O Ni ([NiL] ),
1
7
27
7
3
2
17.58; found, 217.57. UV-vis absorption spectrum (250 μM; 20 mM HEPES, 100 mM NaCl at
-1
-1
-1
pH 7.4, 25 °C): 264 nm (ɛ = 3600 M cm ). FT-IR (KBr, cm ): 3378 (s, broad); 3292 (s); 2927
w); 1667 (s); 1606 (m); 1462 (m); 1422 (m); 1304 (m); 1093 (m); 668 (w); 634 (w).
(

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DOI: 10.1039/C9DT00747D
Preparation of 4% agarose Gel
The 4% agarose gel solution was prepared by following the same procedure as reported
4
8
previously. Distilled water (24 ml) was taken in an erlenmeyer flask, then agarose powder (1.0
g) was added portion wise while stirring vigorously at room temperature. The resulting solution
was heated to boil until the agarose powder was completely dissolved and the solution became
clear. Finally the solution was weighed and appropriate amount of hot distilled water was added
to maintain the original mass.
X-ray structure determination
.
.
Single crystals of [Co(TPTA)] Cl 2H O were obtained by vapour diffusion of diethyl ether over
2
2
.
.
the solution of the complex in MeOH and those of nickel [Ni(TPTA)Cl] Cl 0.5H O were
2
2
2
obtained by slow evaporation of a solution of the complex in a mixture of MeOH and DMF.
Suitable single crystals were selected, coated with paraffin oil and mounted on glass fibres. X-ray
diffraction data were collected at 110.0 (2) K on a Bruker Kappa APEX III Charge-Coupled Device
(CCD) area-detector diffractometer controlled by the APEX3 software package using graphite-
monochromated Mo-Kα radiation (λ = 0.71073 Å) and equipped with an Oxford Cryosystems
Series 700 cryostream. Diffraction images were processed with the software SAINT-Plus and
4
9, 50
absorption corrections were applied using program SADABS.
The structures were solved by
2
direct methods and refined by full-matrix least squares methods on F using the SHELXTL
5
1
package. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were introduced
in calculated positions and refined on a riding model with isotropic thermal parameters twenty
percent larger than the Ueq of the attached non-hydrogen atom. Crystal data and the details of
structure refinement are provided in Table 2.
.
.2H
Table 2 Crystal data, collection, and structure refinement parameter for [Co(TPTA)] Cl
2
2
O and
.
.
[
2 2 2
Ni[TPTA)Cl] Cl 0.5H O
.
.2H
2 2 2
[Ni(TPTA)Cl] O
.Cl .0.5H
C H55 Cl N14 Ni O
34 4 2 6.5
1023.14
110(2)
0.71073
Parameters
[Co(TPTA)] Cl
Co N
543.32
110(2)
0.71073
2
2
O
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
C
17
H
31 Cl
2
7
O
5
Crystal system, space gr.
a (Å)
b (Å)
Triclinic, P -1
9.0833(13)
9.9973(15)
14.468(2)
103.039(7)
93.097(7)
97.532(6)
1264.1(3)
2, 1.427
0.930
566
0.3 x 0.2 x 0.1
2.265 to 28.696°
Triclinic, P -1
11.1550(9)
14.4670(11)
16.4988(12)
65.327(3)
71.425(3)
78.726(3)
2287.4(3)
2, 1.485
C (Å)
α (deg)
β (deg)
γ (deg)
3
Volume (Å )
3
Z, Calculated density (g/cm )
-1
Absorption coefficient (mm )
F(000)
1.116
1066
3
Crystal size (mm )
0.4 x 0.2 x 0.2
2.138 to 28.282°
-14≤h≤14, -19≤k≤19, -21≤l≤21
Theta range for data collection
Limiting indices
-12≤h≤12, -13≤k≤13, -19≤l≤19
Reflections collected / unique
58525 / 6432 [R(int) = 0.1136]
94838 / 11286 [R(int) = 0.0476]

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DOI: 10.1039/C9DT00747D
Completeness to theta
Absorption correction
Refinement method
Data / parameters
99.9 %
Empirical
Full-matrix least-squares on F
6432 / 289
1.163
99.5%
Empirical
Full-matrix least-squares on F
11286 / 547
1.040
2
2
2
Goodness-of-fit on F
Final R indices [I>2σ (I)]
R indices (all data)
Largest diff. peak and hole (e.Å )
R1 = 0.1013, wR2 = 0.3153
R1 = 0.1463, wR2 = 0.3545
1.511 and -1.075
R1 = 0.0526, wR2 = 0.1702
R1 = 0.0638, wR2 = 0.1815
1.874 and -0.799
-3
1
2
/2
2
[
|
푅1 = ∑ 퐹표
|
―
|
퐹푐
|
]
/∑
|
퐹표
|
, 푤푅2 = [∑[푤
(
|
퐹표|2
―
|
퐹푐|2
)
]/∑[푤
(
|
퐹표|2
]]
)
Determination of solution magnetic moment
Evans method 52, 53 was used to calculate the effective magnetic moments of complexes. In order
to determine the magnetic moments of the complexes at different pH, (3-6 mM) of complex was
dissolved in a buffer solution containing 20 mM HEPES, 100 mM NaCl, 5% (v/v) tert-butanol and
1
0% (v/v) D O. The pH of all solutions were maintained to the exact value by addition of 0.5 N
2
HCl and NaOH respectively. The resulting solution was placed in a sealed capillary tube inside
the NMR tube containing same solvent without paramagnetic complex, as a reference solution.
1
Magnetic susceptibility of complexes at variable pH were calculated by H NMR spectra on a
Bruker AVANCE III 400 MHz spectrometer at 310 K (T). Diamagnetic corrections were carried
5
4
based on the empirical formula using Pascal’s constant. The effective magnetic moments (μ )
eff
was calculated using the following equation (1):
[
휒 (푑0 ― 푑 ]
0
푠
―
3훥휈
0
휒푔 =
+ 휒 +
(1)
0
4
휋휈0
푚
In this equation Δν is the difference of frequency (Hz) observed for tert-butanol in the sample
and the reference solutions. 휈 is the spectrometer frequency (Hz), m is the mass of the substance
0
3
-6
3
per cm of solution and 휒 is the mass susceptibility of solvent H O (-0.72 x 10 cm /g for the
0
2
dilute aqueous tert-butanol). The last term is neglected here due to minimal contribution of mass
susceptibility.
Dissociation of complex
1H NMR spectroscopy was used to monitor the stability of complexes under acidic condition, in
presence of biologically relevant ions and other competing transition metal cations. To assess the
+
2
stability of [Co(TPTA)] in acidic condition, sample contained 10 mM complex, 100 mM NaCl,
3-5) mM 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt as a standard at pD 3.6-4.3. For
(
studies in the presence of biologically relevant ions, sample contained 10 mM complex, 100 mM
NaCl, 25 mM K CO , 0.40 mM Na HPO and 5 mM of 3-(trimethylsilyl)-1-propanesulfonic acid
2
3
2
4
sodium salt as a standard at pD 7.4-8.0. For studies with other metal cation, sample contained 10
mM ZnCl , 100 mM NaCl, 5 mM of 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt as a
2
standard at pD 6.8-7.1. All the samples were kept in an incubator at 37 °C and monitored for 72 h.
The pD of all solution were maintained to the exact value by addition of dilute DCl and/or NaOD.

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Electrochemical measurement
Cyclic voltammetric measurement was carried out by using CHI 620D electrochemical analyzer
at ambient temperature under nitrogen atmosphere. The cell was equipped with a glassy carbon
working electrode, a platinum wire as a counter electrode, and an Ag/AgCl as a reference electrode.
The measurement was carried out in aqueous solution with 1 mM complex, 20 mM HEPES, 100
-1
mM NaCl at pH 7.4 with a scan rate 100 mV s . The observed potentials with respect to Ag/AgCl
electrode were converted and referenced to the normal hydrogen electrode (NHE) by using
5
5
standard reported literature conversion factor.
NMR spectroscopy
1
13
H and C NMR spectra of Ligand and its intermediates were recorded at 25 ºC at 400 MHz and
00 MHz frequencies, respectively using Bruker AVANCE III 400 MHz spectrometer. Variable-
1
1
1
1
temperature H NMR spectra and H- H-COSYspectrum of complexes were recorded on the same
spectrometer. H NMR spectra of 10 mM solution of complexes at various pH were recorded at 37
1
ºC in presence of 20 mM HEPES and 100 mM NaCl. The pH of all the solutions were maintained
at the preferred value by addition of 0.5 N HCl and NaOH. For analysis and processing of the
recorded spectra, Topspin 3.5pl7 and MestReNova 10.0 NMR data processing software were used.
Lock was achieved by placing one sealed glass capillary containing D O inside the 5 mm NMR
2
tube.
CEST experiment
CEST experiments were performed on a 9.4 Tesla 400 MHz Bruker AVANCE-III Nanobay NMR
spectrometer using a broadband liquid state probe-head. CEST z-spectra were obtained by plotting
normalized (100 x M /M ) water signal intensities as a function of the presaturation pulse
z
0
frequency offset from the bulk water. Spectra were collected with presaturation at 1 ppm interval
with respect to the bulk water resonance position. Presaturation was achieved at a power level of
2
5 μT with duration of 2 s pulse. Spectra of 10 mM complexes were collected at 37 ºC in the
presence of 20 mM HEPES and 100 mM NaCl. To monitor the efficiency of the complex as a
CEST agent in biological media, separate 10 mM solutions of the complexes were prepared in
rabbit serum, fetal bovine serum (FBS) and agarose gel. All spectra were recorded at 37 °C. To
determine the CEST spectra in agarose gel, solution was prepared by diluting 1 ml of 20 mM
complex, 40 mM HEPES and 200 mM NaCl in 1 ml of 4 % agarose gel solution and finally
adjusting the pH to 7.4. The pH values of the buffer solution, rabbit serum, fetal bovine serium
(FBS) and agarose gel samples were adjusted using 0.5 N HCl and NaOH solution. Lock was
achieved by placing one sealed glass capillary containing D O inside the 5 mm NMR tube.
2
Determination of Exchange Rate Constant
NMR Exchange rate constant of [Co(TPTA)]+2 complex in the four media listed above was
determined by measuring the water signal intensities immediately after on-resonance (푀 ) and off-
푧
resonance (푀 ) irradiation of 4 s to ensure the complete saturation at different pre-saturation power
0

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DOI: 10.1039/C9DT00747D
levels (퐵 ) ranging between 7.5 T to 25 T. The calculation of exchange constant (푘 ) was
1
푒푥
5
6
performed following the procedure described by Dixon et. al. The expected linear relationship
푀푧
1
2
between the quantity
(푀 ― 푀 ) and 휔 was fitted in Microsoft Excel with high correlation
0 푧 1
coefficient of 0.998. 휔 is given by 훾퐵 , 훾 being the gyro-magnetic ratio of proton. The exchange
1
1
1
2
1
2
1
2
constant was then calculated from the intercept on the 휔 axis as 푘 = ―
휔
1
1
푒푥
T relaxivity
1
Longitudinal relaxation rate constant for water protons both in absence and presence of
+
2
[
Co(TPTA)] complex was measured using the inversion recovery method. Relaxation of
inverted bulk water magnetization was monitored as a function of 8 decay intervals ranging from
ms to 10 s. Long repetition time (recycle delay) of 5 s was employed to ensure complete
6
longitudinal relaxation between two measurements. The relaxivity was calculated as the
concentration normalized change in longitudinal rate constant of water in presence of the complex
from that of free water (in absence of the complex).
Cell culture and cytotoxicity
HEK 293 WT were maintained in culture flasks in DMEM supplemented with 10% heat-
inactivated FBS (Gibco), antibiotic cocktail (1 mM penicillin, 1 mM streptomycin) and 2 mM L-
glutamine. Cells were cultured and maintained in a humidified atmosphere at 37°C and 5% CO2.
Cytotoxicity was measured by the MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
5
7
tetrazolium bromide) method as described earlier. Cells (100 μl) were seeded (concentration 5-
5
7
ꢀ×ꢀ10 cells/mL) into a microtitre plate and incubated for 24 h to allow them to adhere.
[
+
2
Co(TPTA)] complex was dissolved in double distilled water to a stock solution of 200 mM.
Further, working dilutions were made tn the concentration range of 20 to 200 mM and 20μl of
diluted compound was added to 400μl media such that the final concentration would range from 1
mM to 10 mM respectively. The negative control wells included cells exposed to 20 μl ddH O.
2
Post compound addition, the microtitre plate was incubated for additional 24 h and the cytotoxitiy
was assayed using the thiazolyl blue tetrazolium bromide (MTT) colorimetric assay. The assay is
based on the ability of live cells to reduce the water soluble MTT into an insoluble formazan
product. Briefly, after a 24 h incubation, 10μl of MTT (5 mg/ml) was added to the cells and
incubated for 5 h. Absorbance of the developed colour was spectrophotometrically determined
using a multi-well plate reader which measured the optical density at 570 nm with a reference
wavelength of 655 nm. Data expressed as mean ± standard error (SE) for each experimental group
were analyzed by one way analysis of variance (ANOVA) using GraphPad Prism statistical
software package version 7.0 (San Diego, USA).
Conflicts of interest
The authors declare no conflict of interest.

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Acknowledgements
AKS acknowledges IIT Bhubaneswar for infrastructure. RNP thanks UGC, Delhi for his
fellowship. AG acknowledges NISER (DAE) for infrastructure. SC acknowledges NISER for the
fellowship. PB thanks UGC, Delhi for Junior and Senior research fellowship.
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For Table of Contents Entry
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Co(II) and Ni(II) complexes of a macrocyclic triamide ligand act as paraCEST MRI contrast
agent in HEPES buffer, FBS, rabbit serum and 4% agarose gel (w/w).