Gear up for a pH shift: A responsive iron(II) 2-amino-6-picolyl-appended macrocyclic paraCEST agent that protonates at a pendent group

Article abstract of DOI:10.1021/acs.inorgchem.6b02159

Two high-spin Fe(II) and Co(II) complexes of 1,4,7,10-tetraazacyclododecane (CYCLEN) appended with four 2-amino-6-picolyl groups, denoted as [Fe(TAPC)]²⁺ and [Co(TAPC)]²⁺, are reported. These complexes demonstrate C₂-symmetrical geometry from coordination of two pendents, and they are present in a single diastereomeric form in aqueous solution as shown by ¹H NMR spectroscopy and by a single-crystal X-ray structure for the Co(II) complex. A highly shifted but low-intensity CEST (chemical exchange saturation transfer) signal from NH groups is observed at -118 ppm for [Co(TAPC)]²⁺ at pH 6.0 and 37 °C. A higher intensity CEST peak is observed for [Fe(TAPC)]²⁺, which demonstrates a pH-dependent frequency shift from -72 to -79 ppm at pH 7.7 to 4.8, respectively, at 37 °C. This shift in the CEST peak correlates with the protonation of the unbound 2-amino-6-picolyl pendents, as suggested by UV-vis and ¹H NMR spectroscopy studies at different pH values. Phantom imaging demonstrates the challenges and feasibility of using the [Fe(TAPC)]²⁺ agent on a low-field MRI scanner. The [Fe(TAPC)]²⁺ complex is the first transition-metal-based paraCEST agent that produces a pH-induced CEST frequency change toward the development of probes for concentration-independent imaging of pH.

Full text of DOI:10.1021/acs.inorgchem.6b02159

Article
pubs.acs.org/IC
Gear Up for a pH Shift: A Responsive Iron(II) 2‑Amino-6-picolyl-
Appended Macrocyclic paraCEST Agent That Protonates at a
Pendent Group
Pavel B. Tsitovich,† Jordan M. Cox,† Joseph A. Spernyak,‡ and Janet R. Morrow*,†
†Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, New York 14260, United States
‡Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, United States
S
* Supporting Information
ABSTRACT: Two high-spin Fe(II) and Co(II) complexes of
1,4,7,10-tetraazacyclododecane (CYCLEN) appended with
four 2-amino-6-picolyl groups, denoted as [Fe(TAPC)]2+ and
[Co(TAPC)]2+, are reported. These complexes demonstrate
C2-symmetrical geometry from coordination of two pendents,
and they are present in a single diastereomeric form in aqueous
1
solution as shown by H NMR spectroscopy and by a single-
crystal X-ray structure for the Co(II) complex. A highly shifted
but low-intensity CEST (chemical exchange saturation trans-
fer) signal from NH groups is observed at −118 ppm for
[Co(TAPC)]2+ at pH 6.0 and 37 °C. A higher intensity CEST
peak is observed for [Fe(TAPC)]2+, which demonstrates a pH-
dependent frequency shift from −72 to −79 ppm at pH 7.7 to 4.8, respectively, at 37 °C. This shift in the CEST peak correlates
with the protonation of the unbound 2-amino-6-picolyl pendents, as suggested by UV−vis and 1H NMR spectroscopy studies at
different pH values. Phantom imaging demonstrates the challenges and feasibility of using the [Fe(TAPC)]2+ agent on a low-field
MRI scanner. The [Fe(TAPC)]2+ complex is the first transition-metal-based paraCEST agent that produces a pH-induced CEST
frequency change toward the development of probes for concentration-independent imaging of pH.
information for disease diagnoses and the appropriate choice
of therapy.5−7 The spatial resolution provided by MRI is
essential to map the alterations of pH that occur at the
extracellular microenvironment (pHe). Several Gd(III)-based
MRI agents utilizing T1- and T2-weighted contrast have been
reported,8−10 including ones based on various nanoparticle
scaffolds.11−13 MRI has certain advantages over other imaging
modalities used in pH measurements, such as electron
paramagnetic resonance (EPR),14,15 positron emission tomog-
raphy (PET),16 and in vivo optical imaging, in terms of tissue
depth penetration, spatial resolution, and availability of
instrumentation in the clinic.17,18
Our laboratory is involved in the development of para-
magnetic transition metal ion complexes as responsive probes
for MRI and MRS (magnetic resonance spectroscopy).19,20 In
particular, CEST MRI is of special interest due to the intrinsic
responsiveness of the exchangeable protons of the contrast
agent to the environment.21 These exchangeable protons are
key to producing contrast through decreasing the water proton
resonance intensity.22,23 Transition metal complexes that have
applications as paramagnetic CEST (paraCEST) agents may
utilize many types of exchangeable protons, including those on
INTRODUCTION
■
Transition metal ion based MRI contrast agents are under
development as alternatives to contrast agents that contain
Ln(III) ions.1,2 Transition metal ions have a rich coordination
chemistry that facilitates the design of new types of responsive
contrast agents. The research presented here describes an
unusual Fe(II) coordination complex that undergoes pH-
dependent structural changes to produce a pH-induced shift of
an exchangeable NH proton. This shift gives rise to a change in
the frequency of the chemical exchange saturation transfer
(CEST) peak. This is the first example, to the best of our
knowledge, of a transition metal complex that produces a shift
in CEST signal as a response to changes in pH. Such an
approach is promising, given that frequency-responsive MRI
probes have certain advantages over intensity-responsive
contrast agents. In particular, a frequency-responsive contrast
agent does not require a correction for tissue concentration of
the probe, unlike a contrast agent that responds by an increase
or decrease in signal.
MRI contrast agents which are responsive to pH in the
biological environment may fill a need in clinical diagnosis.
MRI-based pH measurements are important for elucidation of
acidosis conditions in solid tumors, as well as for renal tubular
acidosis that is indicative of kidney malfunction.3,4 As a result,
mapping of tissue pH by MRI may provide valuable
Received: September 8, 2016
© XXXX American Chemical Society
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Scheme 1. Six-Coordinate Fe(II) and Co(II) Complexes
nitrogen-containing heterocyclic donors, such as pyridines,24
benzimidazoles,19 and pyrazoles.25 Notably, the ionization
properties of imidazole-based compounds have also been
utilized in diamagnetic CEST pH-responsive imaging.26
The aminopicolyl functionalities reported here are members
of a broad class of largely unexplored ligand donors with the
potential for preparing new paraCEST contrast agents for
imaging. A few previous reports demonstrated that amino
groups produce CEST peaks for Ln(III) complexes despite
their weak coordination affinity.27,28 Recent work, however,
showed that there was no CEST effect when an appended
carbamate was reduced to an aminopicolyl pendent in a Ln(III)
complex.29
The sensitivity of chemical exchange to physiological
conditions such as pH makes paraCEST agents amenable for
the development of responsive or “smart” agents.30,31 In this
regard, the rate constants (kex) for the exchange of protons of
the paraCEST agent and bulk water typically vary with pH. The
corresponding change in the intensity of the CEST signal with
pH can be used to measure pH. For example, amide-appended
Ln(III) and paramagnetic transition metal(II) complexes show
the higher CEST intensities with increasing pH, consistent with
a base-catalyzed exchange mechanism.32,33 Alcohol-appended
Ln(III) and Fe(II) complexes demonstrate an increase in
CEST peak intensity also from base-catalyzed exchange.33,34
Utilization of Ln(III) complexes containing two types of
exchangeable protons allows for ratiometric pH sensing.32 In
these probes, the intensity of the amide CEST effect increases
at more basic pH, while the water-based CEST effect is pH
independent.35 A second example of a ratiometric pH probe
based on two sets of different amide and amine NH protons
produces CEST peaks at only |Δω| ≤ 10 ppm from bulk
water.36 The small shift is a significant drawback, since the
CEST signals are obscured by a magnetization transfer (MT)
effect in tissue when they are close to the bulk water frequency.
All of these examples feature CEST agents that undergo a
change in the intensity of the CEST effect with pH.
To produce the first example of a pH responsive transition
metal paraCEST agent that changes CEST frequency with pH,
we designed the TAPC ligand, which contains exchangeable
amino groups in close proximity to the metal ion center
(Scheme 1). As we previously reported, NH2 groups of 3-
amino-2-methyl-6-picolyl pendents produce a strong CEST
effect in a paramagnetic [Fe(AMPT)]2+ complex based on
1,4,7-triazacyclononane (TACN) (Scheme 1).24 However, the
paraCEST signal of this complex is only shifted 6.5 ppm from
bulk water. This small paramagnetically induced proton shift is
consistent with the large distance separation between the amino
group and the metal ion center, which is estimated to be >5.2
Å, as obtained for the Fe2+−H distance of the [Fe(MPT)]2+
crystal structure.40 Here, we present a Fe(II) CYCLEN-based
complex, denoted as [Fe(TAPC)]2+, containing an amino
functionality incorporated into the 2-amino-6-picolyl pendents.
The amino groups of bound picolyl donors flank the metal
binding site, placing exchangeable protons in close proximity to
the paramagnetic center at 3.5 Å as discussed here. Notably,
this paraCEST agent is based on Fe(II), a biologically relevant
transition metal ion, which holds promise as a contrast agent
for imaging. A second analogue based on Co(II) was also
prepared.
EXPERIMENTAL SECTION
■
Syntheses of 6-methyl-2-phthalimidopyridine (II) and 6-bromometh-
yl-2-phthalimidopyridine (III) were reported before,41,42 and the
optimized procedures are described in the Supporting Information.
Phthalimide-Protected TAPC (IV). The bromo derivative III (3.9
g, 12 mmol, 4.2 equiv) was added to an argon-purged solution of
CYCLEN (0.5 g, 2.9 mmol, 1.0 equiv) and DIPEA (3.0 mL, 17 mmol,
6 equiv) in acetonitrile (120 mL), and the reaction mixture was
refluxed for 3 h (Scheme 1). The precipitate that formed upon cooling
to room temperature was filtered and washed with 40 mL of
acetonitrile. The solids were suspended in 30 mL of water, the mixture
was sonicated for 10 min, and a beige precipitate of IV was collected
by centrifugation and dried under vacuum. Yield: 2.5 g, 2.2 mmol
1
(77%). H NMR, 500 MHz (DMSO-d6, ppm): δ 8.01−7.94 m (8H,
Ar), 7.94−7.88 m (8H, Ar), 7.80 t (4H, J = 8 Hz, Ar), 7.76 d (4H, J =
8 Hz, Ar), 7.35 d (4H, J = 8 Hz, Ar), 3.63 s (8H, 4CH2), 2.76 s (16H,
8CH2). 13C NMR, 75 MHz (DMSO-d6, ppm): δ 166.47, 160.38,
144.91, 138.60, 134.89, 131.35, 123.58, 122.81, 120.87, 60.29, 52.86.
ESI-MS (m/z): calculated, 1117.4 [M + H+]; found, 559.5 (15%) [(M
+ 2H+)/2], 1117.5 (100%) [M + H+], and 1139.5 (20%) [M + Na+].
TAPC Ligand. The phthalimide-protected macrocycle IV (0.91 g,
0.81 mmol) was suspended in 120 mL of ethanol, and 1.2 mL of
hydrazine monohydrate (1.2 g, 24 mmol) was added (Scheme 1). The
reaction mixture was refluxed for 5 h. After it was cooled to room
temperature, the reaction mixture was filtered, and the filtrate was
concentrated in vacuo. The crude residue was dissolved in a minimal
volume of an acetonitrile/water (50/50, v/v) mixture and recrystal-
lized in the refrigerator. The crystals were collected and redissolved in
The first example of a paraCEST agent (Δω ≥ 50 ppm) that
demonstrates a pH-responsive change in the CEST frequency
(∼5 ppm) is based on a Eu(III) complex appended with an
ionizable phenol-containing arm.37 The responsiveness of this
agent is due to the deprotonation of phenol at pH values higher
than 6.5, accompanied by isomerization to a quinone-like
conformation. The CEST frequency shift attributed to the
water ligand is most significant at pH 6.0−7.6, making this
agent optimal for biological imaging.38 A Tb(III) analogue with
ionizable phenol gives a CEST peak at −550 ppm, a frequency
which is well beyond the tissue MT effect.39
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Scheme 2. Synthesis of TAPC Ligand and [M(TAPC)]Cl2 Complexes (M2+ = Fe2+, Co2+)
a minimal volume of a methanol/water (30/70, v/v) mixture, and the
acidity of the solution was adjusted to pH 3.5−4.0. Slow rotary
evaporation gave a brown precipitate, which was discarded. Further
evaporation afforded white solids of the TAPC ligand (8HCl salt).
picolyl groups (Scheme 1). In addition, the pH dependence of
the parent [Fe(TMPC)]2+ 1H NMR resonances suggested that
an analogous frequency shift in the exchangeable proton
resonance would be observed in the new TAPC complexes.
The synthetic route for TAPC is presented in Scheme 2. The
phthalimide protection of the amino group was utilized prior to
the bromination of methyl with NBS, followed by alkylation of
CYCLEN and subsequent deprotection with hydrazine, which
resulted in the TAPC ligand in good yield. This phthalimide
protection strategy, which utilizes gentle reaction conditions, is
beneficial in comparison to the pivaloyl-protecting approach,
which requires harsh deprotection conditions.43 The TAPC
complexes were readily formed in methanol/water (1/1)
solutions according to a previously reported procedure.40 The
detailed synthetic protocols are described in the Experimental
Section.
1
Yield: 0.32 g, 0.36 mmol (44%). H NMR, 500 MHz (D2O, ppm): δ
7.84 t (4H, J = 8 Hz, Ar), 7.05−6.95 two d’s (8H, Ar), 4.25 s (8H,
4CH2), 3.37 s (16H, 8CH2). 13C NMR, 75 MHz (D2O, ppm): δ
156.23, 143.32, 142.83, 114.50, 112.61, 55.10, 49.02. ESI-MS (m/z):
calculated, 597.4 [M + H+]; found, 299.4 (100%) [(M + 2H+)/2],
597.4 (95%) [M + H+], and 619.5 (70%) [M + Na+].
Syntheses of Complexes. [Fe(TAPC)]Cl2 and [Co(TAPC)]Cl2
have been prepared in a procedure similar to one previously reported
for the TMPC complexes.40 In a typical procedure, TAPC (0.1 mmol)
was dissolved in a mixture of acetonitrile (0.4 mL) and water (0.8 mL)
at pH below 4.0 followed by addition of 1 equiv of Fe(II) or Co(II)
salt. Small aliquots (1.0−2.0 μL) of 1.0 M NaOH aqueous solution
were gradually added with constant stirring to adjust to pH 7.0 over 1
h. The solid impurities were removed by centrifugation, and solutions
were dried using a SpeedVac centrifugal evaporator. [Fe(TAPC)]Cl2
was obtained as a light beige powder (85% yield). ESI-MS: m/z 326.4
(100%) [m/2], 651.3 (50%) [m − H+], 687.2 (35%) [m + Cl−].
[Co(TAPC)]Cl2 was obtained as a light blue powder (75% yield).
ESI-MS: m/z 327.9 (60%) [m/2], 654.3 (100%) [m − H+], 690.2
(20%) [m + Cl−].
Structural Properties of Complexes in Solution. The
hyperfine-shifted proton resonances of [Fe(TAPC)]2+ span the
range from −75 to +290 ppm and demonstrate narrow line
widths of 50−700 Hz (25 °C) in deuterium oxide, as shown in
Figure 1. Together with the effective magnetic moment of μeff =
1
5.0 0.1 μB, this H NMR spectrum indicates that the TAPC
1
ligand stabilizes the high-spin Fe(II) state. Similarly, the H
RESULTS AND DISCUSSION
NMR resonances of [Co(TAPC)]2+, μeff = 5.2 0.2 μB, are
■
Ligand Design and Synthesis. The TAPC ligand was
designed to stabilize high-spin divalent Fe(II) and Co(II)
complexes, to contain exchangeable NH protons, to produce
symmetrical complexes with low structural fluxionality, and to
provide kinetically inert complexes. The TAPC ligand was
inspired by TMPC, a derivative of 1,4,7,10-tetraazacyclodode-
cane (CYCLEN) containing four 6-methyl-2-picolyl pendents.
TMPC produces six-coordinate, rigid, and redox-stable Fe(II)
and Co(II) complexes, which are shown in Scheme 1.40 These
paramagnetic complexes demonstrate hyperfine shifted proton
resonances of the ligand and effective magnetic moments (μeff)
of 5.7 0.1 and 6.0 0.3 μB in aqueous solution, which are
indicative of high-spin Fe(II) and Co(II) ions, respectively. The
methyl substituents flanking the pyridine N-donor groups in
1
these Fe(II) and Co(II) ions give highly shifted H NMR
resonances at −49.3 and −113.7 ppm, respectively, at 37 °C.40
Exchangeable amine groups were placed at the same
substitution site of pyridine to gain significant dipolar shift
contributions. The TAPC ligand thus contains four 2-amino-6-
1
Figure 1. H NMR of [Fe(TAPC)]2+ in D2O, pD 6.8, at 25 °C. The
inset shows the expanded diamagnetic region. The solvent peak is
labeled “S”.
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Table 1. Crystal Data and Structure Refinement Details for [Co(TAPCH)](CoCl4)Cl(H2O)2
empirical formula
C32H49Cl5Co2N12O2
formula wt
928.94
temp/K
90
cryst syst
monoclinic
space group
P21/n
a/Å
11.7544(5)
b/Å
14.9885(6)
c/Å
23.5529(8)
α/deg
90
β/deg
93.7293(12)
γ/deg
V/Å3
90
4140.8(3)
Z
4
ρcalc/g cm−3
1.490
μ/mm−1
1.170
F(000)
1920.0
cryst size/mm3
0.04 × 0.02 × 0.02
Mo Kα (λ = 0.71073 Å)
3.222−52.77
−14 ≤ h ≤ 10, −18 ≤ k ≤ 18, −28 ≤ l ≤ 28
31091
radiation
2θ range for data collection/deg
index ranges
no. of rflns collected
no. of indep rflns
no. of data/restraints/params
goodness of fit on F2
final R indexes (I ≥ 2σ(I))
final R indexes (all data)
largest diff peak, hole/e Å−3
8400 (Rint = 0.0469, Rσ = 0.0472)
8400/0/487
1.055
R1 = 0.0524, wR2 = 0.1368
R1 = 0.0694, wR2 = 0.1440
0.72, −1.20
dispersed from −110 to 330 ppm (Figure S1 in the Supporting
Information). Moreover, a comparison of the 1H NMR spectra
of the complexes [Co(TAPC)]2+ and [Co(TMPC)]2+, as well
as [Fe(TAPC)]2+ and [Fe(TMPC)]2+, clearly indicates their
structural resemblance in solution due to their similar
spectroscopic properties (Figures S1 and S2). The [Fe-
(TAPC)]2+ complex has 18 nonexchangeable proton reso-
nances of equivalent integrated intensities. Fourteen of these
nonexchangeable proton resonances exhibit significant hyper-
fine shifts, while four proton resonances are found within the
diamagnetic region of the spectrum (Figure 1). This is similar
to the case for the [Fe(TMPC)]2+ complex, which produces 20
nonexchangeable proton resonances, 18 of which have the same
integrated intensities and two demonstrate 3-fold higher
intensities corresponding to the methyl groups (Figure S2).
The presence of one major set of proton resonances indicates
that [Fe(TAPC)]2+ is stabilized in a single diastereomeric form
in aqueous solution. As we have recently shown, the parent
[Fe(TMPC)]2+ complex has a six-coordinate C2-symmetrical
geometry present in a single diastereomeric form.40 On the
basis of a comparison of the solution properties of the two
complexes together with the solid-state structure of [Fe-
(TMPC)]2+, we propose that [Fe(TAPC)]2+ also adopts a six-
coordinate C2-symmetrical geometry. In this geometry, the
Fe(II) ion is coordinated to all four nitrogen donors of the
CYCLEN macrocycle and two picolyl nitrogen donors. Two 2-
amino-6-picolyl groups bound to the metal ion are attached at
the 1,7-amino groups of CYCLEN, and these groups are
expected to bind in a cis configuration relative to the
macrocycle plane. Two other 2-amino-6-picolyl groups are
not bound to the Fe(II) center, and they experience smaller
paramagnetically induced shifts. These pendents produce four
of the proton resonances that are in the diamagnetic region, as
shown in the inset of Figure 1. However, the diastereotopic
nature of the methylene group of the pendent suggests that
there should be five resonances for the unbound pendents,
three on the pyridine ring and two for the methylene. The fifth
proton resonance is assigned as one of the 14 highly
paramagnetically shifted proton resonances. The 1H NMR
spectrum of [Co(TAPC)]2+ is also consistent with a single
diastereomeric C2-symmetrical structure in aqueous solution
(Figure S1A).
Crystal Structure. The Co(II) complex cation crystallized
in the centrosymmetric P21/n space group, and the complete
crystal data and structure refinement parameters are given in
Table 1 and in the Supporting Information. In keeping with the
symmetry of the unit cell, the enantiomeric form of the
complex is present in the lattice as well. The complex cation has
a total charge of +3 formed by Co2+ and H+ from one
protonated pyridine atom ([Co(TAPCH)]3+). Counterions
include the [CoCl4]2− anion and one chloride ion. Notably, the
pyridinium ion is formed on a single unbound 2-amino-6-
picolyl group.
The complex cation of [Co(TAPCH)]3+ is six-coordinate
with four CYCLEN amine donors and two pyridine nitrogen
atoms (Figure 2). Two Co(II)-bound 2-amino-6-picolyl
pendents at the 1,7-nitrogen atoms of CYCLEN are in a cis
configuration relative to the macrocycle plane. The complex
cation demonstrates an approximately C2 symmetrical structure,
if the pyridinium proton is omitted from consideration. This
geometry is best described as slightly distorted trigonal
prismatic by using the quasi-C3 axis of the trigonal prism,
which is perpendicular to the C2 symmetry axis (Figure 2). The
twist angles for each pair of apical points are calculated and
shown at the bottom of Figure 2. An overlap of [Co-
(TAPCH)]3+ and [Co(TMPC)]2+ crystal structures demon-
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CEST Spectra. CEST contrast is produced by transfer of
selectively saturated spins from the CEST agent, such as
exchangeable NH protons of TAPC, to protons of bulk water.
The bulk water signal decreases upon exchange with the
saturated spins, and this provides a mechanism for producing
an on/off contrast switch controlled by the presaturation pulse
frequency.21 A CEST spectrum is plotted as a percent decrease
in water proton intensity as a function of presaturation pulse
frequency. Both Fe(II) and Co(II) complexes of TAPC
demonstrate highly shifted CEST peaks under slightly acidic
conditions (Figure 3). A 10 mM [Fe(TAPC)]Cl2 solution
Figure 2. ORTEP plots of [Co(TAPCH)]3+ of Λ(δδδδ) configuration.
The hydrogen atoms, counterions, and solvent molecules have been
omitted for clarity, while one pyridine nitrogen atom (N7) is
protonated in the model. Ellipsoids are set at 50% probability. Twist
angles are shown for the projections of two triangles formed by three
donor atoms each when viewed down the quasi-C3 axis of symmetry
perpendicular to the C2 axis of symmetry.
strates their similarity (Figure S3). The nitrogen atoms of the
amines on the bound pendents are 3.46(6) Å away from the
Co(II) center, while for the unbound pendents this distance is
7.62(5) Å. The selected bond lengths and angles are presented
in Table 2. The Co2+−N1, Co2+−N2, and Co2+−N5 bond
lengths are 2.176(3), 2.380(3), and 2.196(3) Å, which are
consistent with the high-spin state of the Co(II) center,
supporting the paramagnetic properties of this complex.40
Figure 3. CEST effect of (A) 10 mM [Fe(TAPC)]2+ at 37 °C and (B)
26 mM [Co(TAPC)]2+ at 37 and 45 °C in 20 mM MES, pH 6.0, 100
mM NaCl, B1 = 24 μT, and 4 s presaturation pulse. The insets show
expanded temperature-dependent CEST effects of 10 mM [Fe-
(TAPC)]2+ and 26 mM [Co(TAPC)]2+.
produced a 14% CEST effect at −74 ppm from bulk water at
pH 6.0 and 37 °C (B1 = 24 μT, 4 s presaturation pulse), as
Table 2. Selected Bond Lengths (Å) and Bond Angles (deg)
for [Co(TAPCH)]3+
1
shown in Figure 3A. A comparison of the H NMR spectra of
[Fe(TAPC)]2+ in D2O and H2O at 25 °C demonstrates
exchangeable protons at −78 ppm from bulk water at 25 °C
(Figure S4). Given the temperature dependence of hyperfine
shifts, these data suggest that the [Fe(TAPC)]2+ CEST peak at
37 °C arises from the NH protons. The [Fe(TAPC)]2+ CEST
peak increases to 41% when the temperature is increased to 55
°C, as shown in the inset of Figure 3A. The [Co(TAPC)]2+
CEST effect is significantly weaker, and it is observed only at
high complex concentrations at elevated temperature, as shown
in Figure 3B. Notably, one highly shifted [Co(TAPC)]2+ CEST
effect (4.4%) is observed at −114 ppm (45 °C), and another
CEST signal is found at 14 ppm from bulk water. This indicates
that two different exchangeable pools are present in this
complex. The highly shifted CEST peak is assigned to the NH
Bond Lengths (Å)
Co1−N2
Co1−N1
Co1−N3
2.380(3)
2.176(3)
2.176(3)
Co1−N5
Co1−N4
Co1−N9
2.196(3)
2.347(3)
2.245(3)
Bond Angles (deg)
N1−Co1−N2
N1−Co1−N4
N1−Co1−N5
N1−Co1−N9
N3−Co1−N1
N3−Co1−N2
N3−Co1−N4
N3−Co1−N5
78.34(11)
78.07(11)
77.38(11)
152.84(11)
123.73(11)
76.59(11)
79.92(11)
151.07(12)
N3−Co1−N9
N4−Co1−N2
N5−Co1−N2
N5−Co1−N4
N5−Co1−N9
N9−Co1−N2
N9−Co1−N4
76.82(11)
128.73(10)
90.58(11)
126.94(11)
91.27(11)
126.96(11)
89.86(11)
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protons of the Co(II)-coordinated 2-amino-6-picolyl pendents,
while the less shifted CEST peak is assigned to the unbound 2-
amino-6-picolyl groups. No enhancement of the [Co-
(TAPC)]2+ CEST signal was found at pH 5.5 or 7.2 (Figure
S5), in comparison to pH 6.0 (Figure 3B). Given the low
intensity of the [Co(TAPC)]2+ CEST peak, further studies
were carried out only on the [Fe(TAPC)]2+ complex.
7.7−6.7. However, under conditions more acidic than pH 6.7,
the CEST peak shifts away from bulk water (Figure 4B) and
the values of Δω change monotonically from −72 ppm at pH
6.7 to −79 ppm at pH 4.8 and 37 °C. Notably, such a sigmoidal
type dependence of CEST on pH (eq S3) indicates an
ionization process in [Fe(TAPC)]2+ (Figure 4B). On the other
hand, two nearly linear regions of the Δω dependence on pH
allow for the linear correlation between them (Figure S6). In
addition, the CEST peak increases in intensity from pH 7.7 to
6.0 and then decreases in intensity as the pH is lowered further
(Figure 4C).
Both the intensity and position of the [Fe(TAPC)]2+ CEST
signal are sensitive to pH, as shown in Figure 4A. No detectable
change in the CEST peak frequency (Δω) is observed at pH
To better interpret these trends, the exchange rate constants
(kex) of the amino protons were determined by using the ω plot
method (Figures S7 and S8).34 A rate constant for proton
exchange at pH 7.7 of 1700 s−1 was obtained. Further
experiments showed that exchange rate constants have a
shallow dependence on pH with an increase in rate constant
from pH 7.7 to 6.0, as shown in Figure S7B. This pH trend is
consistent with acid-catalyzed exchange, as reported previously
for amino groups.28 Notably, the CEST peak intensity
decreases and the peak position shifts as the pH is further
decreased from 6.0 to 4.8. This trend is attributed to the
formation of a new, more highly protonated species. This
protonated species present at acidic pH has been further
studied by using UV−vis spectroscopy and by NMR spectros-
copy, as described below. As a function of temperature, the
exchange rate constant steadily increases as temperature is
increased from 37 °C (1800 s−1) to 55 °C (2800 s−1), as shown
in Figure S8. These data correspond to an increase of CEST
signal at the elevated temperatures (Figure 3A and Figure S9).
Notably, the relatively large chemical shift of the exchangeable
NH protons from bulk water (Δω) allows for a large rate
constant without producing exchange broadening of the CEST
peak.
To further study the effect of pH and different conditions,
the [Fe(TAPC)]2+ CEST effect was studied in buffers
comprised of different ions at various pH values. Notably, the
[Fe(TAPC)]2+ CEST effect was altered only slightly by the
different anions. As shown in Figures S10 and S11, the CEST
effect has similar intensity in MES, HEPES, and phosphate
buffers (PO43−) at similar pH values over a period of 24 h. This
is an important result, because the CEST signal of 10 mM
[Fe(TAPC)]2+ is not significantly affected by 20 mM
phosphate over 12 h, which is present in biological systems
1
in up to 0.4 mM concentrations. Moreover, as shown by H
NMR and CEST studies, [Fe(TAPC)]2+ shows little kinetic
dissociation under acidic conditions of pH 3.4 0.2 (Figure
S12), as well as in the presence of biologically relevant anions,
such as PO43−, CO32−, and Cl− with 16% and 8% dissociation
over 24 h, respectively (Figures S12 and S13 and Table S1).
These data show that the [Fe(TAPC)]2+ complex has some
degree of kinetic inertness, although it is not as inert as an
Fe(II) complex of a CYCLEN derivative that contains four
bound pendents.44
UV−Vis Spectroscopy Studies. In order to better
understand the protonation of the complex as a function of
pH, the UV−visible spectra of the pyridine groups were
monitored. Absorbance spectra of [Fe(TAPC)]2+ solutions at
various pH values are presented in Figure 5A. A broad
absorbance band with a maximum at about 300 nm is assigned
to the pyridine pendent π−π* ligand-centered transitions. No
significant absorbance changes are detected when the solution
is acidified from pH 7.5 to 6.5. When the pH of the solution is
Figure 4. (A) pH dependence of the 10 mM [Fe(TAPC)]2+ CEST.
(B) Dependence of exchange frequency Δω (ppm) of CEST maxima
on the pH of solution. The solid line represents a fit to a sigmoidal
function (eq S3). (C) pH dependence of CEST maxima. Legend: (*)
the maximum values of CEST are taken at various Δω values (ppm)
for pH 4.8 to 7.7. Conditions: 10 mM [Fe(TAPC)]2+, 20 mM
HEPES/MES, 100 mM NaCl, B1 = 24 μT, 4 s presaturation pulse, 37
°C.
F
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Inorganic Chemistry
Article
The pH-dependent UV−vis properties of 2-amino-6-
methylpyridine as a model compound were studied for
comparison. Similar to the case for [Fe(TAPC)]2+, an increase
of absorbance, as well as a red shift from 292 to 304 nm, is
observed when a solution of the model pendent group was
acidified from pH 7.7 to 3.9 (Figure S14A). Fitting of the pH-
dependent absorbance intensity of 2-amino-6-methylpyridine at
λ 304 nm gives a pKa of 7.12
0.06 (Figure S14B).
Interestingly, the difference in the pKa values of a free 2-amino-
6-methylpyridine and the [Fe(TAPC)]2+ unbound pendents is
2.4 pH units. The higher acidity of the protonated pendents of
the complex in comparison to that of the simple amino-
pyridine is consistent with the large cationic charge on the
complex.
NMR Spectroscopy Studies of pH-Induced Structural
Changes. The proton NMR spectrum of [Fe(TAPC)]2+
changes in response to pH, with selective effects on a few
resonances. The line widths (50−700 Hz) and intensities of the
[Fe(TAPC)]2+ proton resonances remain unchanged in D2O
solutions in the range of pD 8.3−6.1 (Figure S15). However, at
more acidic values (pD 5.8−5.1), there is a marked effect on a
few of the paramagnetically shifted resonances, in particular
those in the range of 50−100 ppm and in the diamagnetic
region (Figure S16). These resonances shift or broaden into the
baseline. As shown more clearly in Figure S17 for pD 5.1 versus
pD 7.3, most of the other resonances demonstrate at least some
pH-induced shift (<5 ppm). The four resonances in the
diamagnetic region of the spectrum are also affected by a
decrease in pD at the most acidic values of pD 5.1−6.5 (Figure
S16B). However, chemical shift differences are smaller for these
resonances that have little paramagnetic contribution (ca. 0.5
ppm). These chemical shift differences are analogous to those
observed in the protonation of the model compound 2-amino-
6-methylpyridine at values close to its pKa of 7.71 0.03 in
D2O (Figures S18 and S19).45 The pH dependence of the
resonances is thus most consistent with protonation of the
complex to give minor changes in overall conformation.
Unfortunately, the paramagnetic nature of the complex makes
it difficult to assign proton resonances by using standard NMR
spectroscopy methods. Theoretical calculations that predict the
paramagnetic proton chemical shifts are challenging to do,
given the large contributions from both contact and
pseudocontact shifts, although recent success on Fe(II)
complexes has recently been reported.46 Most notably, the
structural changes of [Fe(TAPC)]2+ in solution are completely
reversible. No oxidation to Fe(III) or dissociation of [Fe-
(TAPC)]2+ is observed by 1H NMR spectra consecutively
taken at pD 8.3, 5.1, and 6.4 (Figure S20). This indicates that
the inner-sphere six-coordinate geometry produced by N-
donors of [Fe(TAPC)]2+ is not disturbed under acidic
conditions. These data and those of the UV−vis spectral
changes are most consistent with protonation of unbound 2-
amino-6-picolyl pendents.
Figure 5. (A) pH dependence of the UV−vis spectra of [Fe-
(TAPC)]2+ and (B) pH dependence of the absorbance intensity of
[Fe(TAPC)]2+ at λ 308 nm with a curve showing a fit of the data to eq
S4 in the Supporting Information that gives pKa = 4.68
0.02.
Conditions: 38 μM [Fe(TAPC)]2+, 2 mM HEPES, 50 mM NaCl in
H2O at 37 °C.
further decreased from pH 6.5 to 2.4, a red shift of the π−π*
absorbance band from 296 to 308 nm is observed, while the
intensity of this absorbance band increases. This pH-dependent
absorbance change is reversible, and there is an isosbestic point
at 281 nm, suggesting a pH-dependent interconversion
between species of different protonation states in solution at
37 °C. Fitting of the pH-dependent absorbance intensity of
[Fe(TAPC)]2+ at λ 308 nm gives a pKa value of 4.68 0.02
(Figure 5B). These data are consistent with the protonation of
the amino-picolyl groups as the source of the CEST peak shift.
These data do not enable us to distinguish which amino-picolyl
groups are protonated. However, the fact that the absorbance
change from pH 6.5 to 4.0 is twice that of a simple 2-amino-6-
methylpyridine model compound for solutions of the same
concentration suggests that there are two amino-picolyl groups
that become protonated over this pH range. Note that it is
unlikely that the unbound picolyl groups would protonate
outside of the pH range studied here. Thus, the good fit of the
titration data to an equation with a single ionization event is
most consistent with the pKa values of the two pendents being
very close to each other. The reversible nature of the
protonation as shown by an absorbance titration also supports
the involvement of the pendent amino-picolyl groups, as it is
difficult to imagine that protonation of the coordinated groups
would leave the complex intact.
Phantom Imaging. Finally, the possibility of using the
CEST peak position for the registration of pH was studied
further for [Fe(TAPC)]2+. Notably, over the pH range of 4.8−
6.7, there is a nearly linear dependence of CEST peak position
with pH, although the shift in position is small (Figure 6A).
While these small chemical shift differences are readily
distinguished on a high-field NMR spectrometer (Figure 4),
analogous studies on a MRI scanner of lower field strength are
more challenging. The phantom images of the [Fe(TAPC)]2+
solutions at pH 4.8, 5.9, and 6.7 were obtained after applying a
G
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Inorganic Chemistry
Article
shape, studies with the Eu(III) complex used a ratio of the
CEST intensity at two different frequencies. The CEST ratio
varied linearly with pH, as shown both on an NMR
spectrometer at 9.4 T and on a high-field MRI scanner at 9.4
T. In contrast, our work suggests that other approaches may be
more viable, especially on low-field MRI scanners. For example,
paraCEST agents with two CEST peaks, each with a different
pH-dependent intensity, were successfully used for ratiometric
registration of pH both at 11.7 T and on a 4.7 T MRI
scanner.47−49 This highlights some of the challenges of using
shift-dependent CEST agents for pH mapping. Notably, the
deconvolution of the CEST shift with both temperature and
pH is another challenge.
SUMMARY AND CONCLUSIONS
■
The versatile chemistry of high-spin Fe(II) facilitates the design
of complexes of different coordination numbers and geo-
metries. Remarkably, even complexes based on the same
tetraaza macrocycle may be either eight-coordinate33,47 or six-
coordinate.50 The coordination sphere of [Fe(TAPC)]2+ with
two bound pendents and two additional pendents provides an
unusual pH response. The position of the CEST peak shifts
monotonically under acidic conditions at values less than pH
6.7. This change in Δω is attributed to subtle structural changes
of the complex, which in turn are caused by protonation of the
unbound 2-amino-6-picolyl pendents of the TAPC ligand, as
supported by UV−vis spectroscopy, by 1H NMR spectroscopy,
and by the observation of the [Co(TAPCH)]3+ complex cation
in the solid state. The structural changes that occur upon
protonation may involve a change in orientation of the
pendents. For example, the protonated 2-amino-6-picolyl
pendents would likely be oriented to avoid any interaction
with the cationic Fe(II) center. Notably, a crystal structure of
an tetrakis(pyridyl)-appended CYCLEN complex of Fe(II) has
two bound pendents and two additional pendents which are
too far from the Fe(II) center to be considered as within a
typical bonding distance but yet are oriented toward the Fe(II)
center.50
The rate constant for exchange of the amino protons of
[Fe(TAPC)]2+ with water is 1900 s−1 at 37 °C and pH 6.0.
This relatively large rate constant is typical of amine groups
including that of an uncoordinated aliphatic NH2 group in a
lanthanide complex (2100 s−1 at pH 7.4)28 and a free amino
group of acetyl-Lys-NH2 (4000 s−1 at pH 7.0).51 For
[Fe(TAPC)]2+, the proton exchange rate constant is nearly
independent of pH over the range of 4.8−7.7 with slightly
higher values at 6.0 that correspond to higher CEST peak
intensity, whereas a modest (2-fold) difference in rate constant
is observed for that of the aliphatic amino group.28 In contrast,
the rate constants for exchange of NH protons of amide
pendents are low, ranging from 300 to 800 s−1 at neutral pH.20
ParaCEST agents with amide pendents generally undergo base-
catalyzed proton exchange with CEST peak intensity and
corresponding exchange rate constants increasing with pH.
The basis for the relatively low intensity of the CEST effect
for both the Fe(II) and Co(II) complexes is not clear at this
juncture. Under similar pH 6.9 conditions at 37 °C, a Co(II)
complex with three pyrazole pendents gives a CEST effect of
24% for three NH protons25 in comparison to the 8% observed
here for [Fe(TAPC)]2+ and 2% for [Co(TAPC)]2+. This is
despite the fact that there are four equivalent exchangeable NH
protons for the last two complexes. For [Co(TAPC)]2+, all
proton resonances are broadened slightly, consistent with either
Figure 6. (A) Dependence of exchange frequency Δω (ppm) of CEST
maxima on pH at 37 °C obtained on a NMR spectrometer at 11.7 T.
The red line represents a linear fit, R2 = 0.978. Colored arrows show
the radio frequencies of interest for imaging pH 4.8, 5.9, and 6.7. (B)
CEST images of phantoms at 37 °C on an MRI 4.7 T scanner.
Solutions of 12 mM [Fe(TAPC)]2+ in 100 mM NaCl and 20 mM
MES buffer of pH 4.8, 5.9, and 6.7 were irradiated at −79, −74, and
−72 ppm. The dotted circles represent the tubes containing (a) water
and (b) 20 mM MES buffer, 100 mM NaCl. (C) Bar graph
representing percent CEST at three pH values when samples are
irradiated at different frequency offsets as in (B).
presaturation pulse at −79, −74, and −72 ppm, respectively
(Figure 6B). The intensity of CEST obtained at 4.7 T by MRI
(Figure 6C) correlates with the shift of the optimal radio
frequency for a given pH at 11.7 T by NMR (Figure 6A).
However, the intensities of phantoms at pH 5.9 and 6.7 are
nearly indistinguishable within error upon saturation at −72
ppm (Figure 6B, C). Thus, no further efforts were made to
study CEST peak position as a function of pH on the MRI
scanner. The difficulty in resolving the change in the CEST
signal with pH is attributed to the poorer precision and spectral
resolution on the 4.7 T MRI scanner.
Interestingly, the pH responsive Eu(III) paraCEST agent
reported by Sherry et al. had a similar CEST peak shift of 6
ppm with pH.37,38 Rather than scan through a range of radio
frequencies for the presaturation pulse to map out the peak
H
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Inorganic Chemistry
Article
a fluxional process or a less than optimal electronic relaxation
time.40 Previous work in our laboratory has shown that
complexes that have broad proton resonances due to either
dynamic processes or electronic relaxation times that are
unfavorable for paraCEST,19 such as found for certain Ni(II)
complexes, produce less intense paraCEST peaks.52 However,
the [Fe(TAPC)]2+ nonexchangeable proton resonances are not
broadened, leading us to suggest that the inherently lower
CEST effect for the amino group, as also observed for a Ln(III)
derivative, is characteristic of this functionality.27−29,36 Efforts
to better understand the factors that contribute to the shape
and intensity of the CEST peak are needed. Notably, the Bloch
equations that have been used to model CEST peak intensity
for Ln(III) paraCEST agents do not take into account
properties such as differences in electronic relaxation times
for different metal ion geometries or dynamic solution
processes that would affect CEST peak intensity.53
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b02159.
Experimental details including NMR spectra, CEST
spectra, Omega plots to obtain exchange rate constants,
synthetic procedures, and crystallographic data (PDF)
Crystallographic data (CIF)
AUTHOR INFORMATION
■
Corresponding Author
*J.R.W.: e-mail, jmorrow@buffalo.edu; fax, 716-645-6963.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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J.R.M. and J.A.S. thank the NIH (CA175858) and J.R.M.
thanks the NSF (CHE-1310374) for support of this work.
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DOI: 10.1021/acs.inorgchem.6b02159
Inorg. Chem. XXXX, XXX, XXX−XXX