Development, characterisation and: In vitro evaluation of lanthanide-based FPR2/ALX-targeted imaging probes

Article abstract of DOI:10.1039/c9dt03520f

We report the design, preparation and characterisation of three small-molecule, Formyl Peptide Receptor (FPR)-targeted lanthanide complexes (Tb·14, Eu·14 and Gd·14). Long-lived, metal-based emission was observed from the terbium complex (τ_H2

Full text of DOI:10.1039/c9dt03520f

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Development, characterisation and in vitro
evaluation of lanthanide-based FPR2/ALX-targeted
imaging probes†
Cite this: Dalton Trans., 2019, 48,
16764
a
Tamara Boltersdorf,a Junaid Ansari,b Elena Y. Senchenkova,b Lijun Jiang,
Andrew J. P. White, a Michael Coogan, c Felicity N. E. Gavins
*
b,d and
a
Nicholas J. Long
*
We report the design, preparation and characterisation of three small-molecule, Formyl Peptide Receptor
(FPR)-targeted lanthanide complexes (Tb·14, Eu·14 and Gd·14). Long-lived, metal-based emission was
observed from the terbium complex (τH O = 1.9 ms), whereas only negligible lanthanide signals were
2
detected in the europium analogue. Ligand-centred emission was investigated using Gd·14 at room
temperature and 77 K, leading to the postulation that metal emission may be sensitised via a ligand-based
charge transfer state of the targeting Quin C1 unit. Comparatively high longitudinal relaxivity values (r1) for
octadentate metal complexes of Gd·14 were determined (6.9 mM−1 s−1 at 400 MHz and 294 K), which
could be a result of a relative increase in twisted square antiprism (TSAP) isomer prevalence compared to
typical DOTA constructs (as evidenced by NMR spectroscopy). In vitro validation of concentration
responses of Tb·14 via three key neutrophil functional assays demonstrated that the inflammatory
responses of neutrophils (i.e. chemotaxis, transmigration and granular release) remained unchanged in
the presence of specific concentrations of the compound. Using a time-resolved microscopy set-up we
were able to observe binding of the Tb·14 probe to stimulated human neutrophils around the cell periph-
ery, while in the same experiment with un-activated neutrophils, no metal-based signals were detected.
Our results demonstrate the utility of Tb·14 for time-resolved microscopy with lifetimes several orders of
magnitude longer than autofluorescent signals and a preferential uptake in stimulated neutrophils.
Received 2nd September 2019,
Accepted 24th October 2019
DOI: 10.1039/c9dt03520f
rsc.li/dalton
The Formyl Peptide Receptor (FPR) family6 comprising
FPR1, FPR2/ALX (also known as the lipoxin A4 receptor, and
Introduction
The inflammatory response is implicated in the progression of previously termed FPR-like 1 [FPRL1, but not to be confused
a variety of disease states including Alzheimer’s disease,1 with FPR1, nomenclature detailed in ref. 6]) and FPR3 (also
stroke,2 sickle cell disease3 and cancer4 and often the extent of known as FPRL2) in humans are categorised as G-protein
inflammation is a key factor controlling disease prognosis. As coupled receptors (GPCR) and have attracted interest as a
a result, there is interest in gaining mechanistic insights into result of their role in modulating both pro- and anti-inflamma-
the physiological processes underlying inflammation in order tory pathways7–9 during all phases of the inflammatory
to address clinical issues and provide screening tools for drug response (including initiation, chemotactic signalling, propa-
discovery programmes.5
gation, resolution and tissue repair). Amongst these, FPR1 and
FPR2/ALX are expressed on the cell surface of human neutro-
phils and can induce chemotaxis, mobilise adhesion mole-
cules and inhibit migration of these immune cells.6 Although
these receptors are also known to be upregulated in a variety
of disease conditions,10 the understanding of their specific
physiological functions still remains limited, creating a need
to prepare probes that enable their roles to be studied in a bio-
logical context.7,11
aDepartment of Chemistry, Imperial College London, Molecular Sciences
Research Hub, White City, London, W12 0BZ, UK. E-mail: n.long@imperial.ac.uk
bDepartment of Molecular & Cellular Physiology, Louisiana State University Health
Sciences Center-Shreveport, Shreveport, LA, 71130, USA.
E-mail: Felicity.Gavins@brunel.ac.uk
cDepartment of Chemistry, Lancaster University, Lancaster, LA1 4YB, UK
dDepartment of Life Sciences, Brunel University London, Uxbridge, Middlesex,
UB8 3PH, UK
Compounds to enable targeting of FPR1 and FPR2/ALX to
track neutrophils in inflammatory conditions have been pre-
viously reported as imaging agents for example, in magnetic
†Electronic supplementary information (ESI) available: Crystallographic data
and supplementary figures. CCDC 1945848. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c9dt03520f
16764 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
resonance imaging (MRI),12 positron emission tomography
(PET),13 optical14 and single-photon emission computed tom-
ography (SPECT).15,16 These constructs, however, have been
derived from the peptide-based FPR1 antagonist cinnamoyl-F-
(D)L-F-(D)L-F (cFLFLF), which is highly hydrophobic and often
results in poor target to background ratios, necessitating incor-
poration of, for instance, large PEGylated chains.17 Another
strategy reports synthetic modifications of the potent, peptide-
based FPR agonist formyl-Met-Leu-Phe (fMLF) to prepare
agents for both optical- and radio-imaging of FPRs.18–20 These
compounds, however, are unselective for receptor subtype
(FPR1 or FPR2/ALX) and the known high potency of the modi-
fied agonist ligand fMLF results in a risk of unwanted receptor
activation,6 possibly even causing an inflammatory response
when using the system for imaging.
To circumvent the issues encountered with peptide
systems, small-molecule, FPR-specific ligands that are syntheti-
cally and metabolically more robust, as well as cheaper to
prepare and modify,21 could provide a convenient alternative.
Large-scale screening for such binding motifs have been pre-
Fig. 1 Schematic illustrating (A) the multiple emission sources occuring
in intensity-based luminescence measurements (from organic probe
component, metal, and diverse autofluorescent sources within a neutro-
phil) and (B) the possibility to separate metal-based lifetime (µs to ms
scale) from all other signals (ns scale).
viously performed, and
a non-peptidic, mild FPR2/ALX
agonist, 4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-
reported using an iridium complex coupled to a peptidic
FPR2/ALX agonist.38 However, the probe was designed based
on a potent FPR2/ALX agonist and caused a dose-dependent
response in human umbilical vein endothelial cells (HUVECs),
altering cellular properties and rendering it not ideal as an
imaging agent.38
2H-quinazolin-3-yl]-benzamide (Quin C1),22 was identified in
2004. The compound was assessed for its concentration depen-
dence in vitro, and in vivo properties were evaluated,22,23 but to
our knowledge it has not been derivatised to form an imaging
agent for FPR2/ALX.
Fluorescence microscopy is a widely used technique for cel-
lular imaging and pre-clinical models that surpasses many
other modalities in terms of sensitivity and spatial resolution,
allowing systems to be investigated on a cellular and subcellular
level.24 Although numerous targeted and fluorescent probes
have been designed to enable visualisation of surface receptors
by fluorescence microscopy, these are often accompanied with
various drawbacks.25 Amongst these are poor photostability,
difficulty in separating signals from autofluorescence, large
probe sizes, as well as sometimes restricted labelling sites when
dealing with peptide-based targeting groups.26,27
Time-resolved microscopy relies on detection of the lumine-
scence lifetimes of a sample, which are not substantially
affected by photobleaching events. In addition, if a sufficiently
long-lived probe is used, this can create signal differences of
several orders of magnitude between compound and cellular
background, allowing for a much more accurate picture of the
agent within a cell.28,29 For example, long-lived iridium probes
have been developed to image cancer cells30,31 and for uses in
live zebrafish.32,33
Therefore, by combining the small-molecule targeting
group Quin C1 with a stable Tb(III) chelate, we aimed to
prepare a probe that does not change the functional response
of the neutrophil, but allows for time-resolved imaging of
these cells (i.e. inflammation) via FPR2/ALX (Fig. 1).
Furthermore, differentiation of compound luminescence from
an autofluorescent background (which is hard to distinguish
in traditional fluorophores) allows for visualisation of com-
pound uptake patterns within the neutrophil in both its acti-
vated state (when prior stimulation has occurred using an
inflammatory stimulus, tumor necrosis factor alpha [TNFα])
and in an un-activated state (using phosphate buffered saline
[PBS] as a control). By complexing Gd(III) instead of Tb(III), we
were also able to prepare a compound with r1 relaxivity values
of 6.9 mM−1 s−1, which are increased compared to typical octa-
dentate structures (where r1 = 3–5 mM−1 s−1).39 The Eu(III) ana-
logue was constructed to enable the macrocyclic conformation-
al equilibria to be investigated by 1H-NMR spectroscopy and
therefore provide insight into the twisted square antiprism
(TSAP) to square antiprism (SAP) geometry ratios of the
complex.
Stable lanthanide(III) chelates based on terbium or euro-
pium often have highly desirable properties for cellular
imaging: their emission is long-lived (up to ms range), the sep-
aration between excitation and emission bands is large and
metal-based emission is characterised by sharp, distinctive
peaks that are only minimally altered by different ligand
surroundings.34–37
Results and discussion
Synthetic procedures
Recently, the first and, to the best of our knowledge, only The FPR2/ALX targeting unit Quin C1 (6) was prepared accord-
example of time-resolved FPR2/ALX imaging in cells has been ing to modified procedures previously described22 and
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16765

View Article Online
Paper
Dalton Transactions
obtained as a crystalline solid after purification by column
chromatography (Fig. 2 and 3). Compound 6 was further
reacted with chloroacetyl chloride to produce 7. Next, a DOTA-
derived, macrocyclic scaffold was prepared following known
protocols.40–42 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra-
acetic acid (DOTA)-derived macrocycles are commonly used for
biomedical applications and their octadentate lanthanide
coordination complexes are characterised by high kinetic inert-
ness and thermodynamic stability under physiological con-
ditions.43 In brief, the mono-tert-butyloxycarbonyl protected
compound 9 was prepared and reacted with chloroacetyl chlor-
ide, before combination with the tert-butyl protected 1,4,7,10-
tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) macrocycle
to yield 11. Subsequent deprotection with trifluoroacetic acid
and dichloromethane unmasked the amine terminal ligand
scaffold 12 in high yields.
Metal complexation was carried out using terbium or euro-
pium trifluoromethanesulfonate salts or gadolinium chloride
hexahydrate in water at pH 5.5 to produce the lanthanide
complexes Tb·13, Eu·13 and Gd·13. The compounds were puri-
fied by dialysis using a 500–1000 Da molecular weight cut-off
cellulose membrane, allowing separation of excess metal salts.
Absence of free lanthanide ions was ascertained using the
xylenol orange indicator test. To afford FPR2/ALX targeted
lanthanide complexes, Tb·13, Eu·13 and Gd·13 were combined
with the functionalised targeting moiety 7. The resulting
compounds Tb·14, Eu·14 and Gd·14 were isolated after
Fig. 3 (A) Crystal structure of 6 (50% probability ellipsoids) and (B)
excerpt of structure showing H-bonding interactions.
Sephadex size exclusion chromatography, dissolved in
a
minimum amount of methanol and precipitated using diethyl
ether.
Fig. 2 Synthetic pathway for preparation of the FPR2/ALX-targeted imaging probes. Reagents: (i) 1-bromobutane, K2CO3, MeOH, (ii) hydrazine
monohydrate, EtOH, (iii) 2-nitro-benzoyl chloride, K2CO3, DCM, (iv) zinc dust, AcOH, DCM, (v) 4-methoxy benzaldehyde, citric acid, EtOH, (vi) chlor-
oacetyl chloride, triethylamine, DCM, (vii) di-tert-butyl dicarbonate, EtOH, (viii) chloroacetyl chloride, triethylamine, DCM, (ix) DO3A, K2CO3, MeCN,
(x) trifluoracetic acid, DCM, (xi) Ln (Ln = Tb, Gd, Eu) trifluoromethane sulphonate, water pH 5.5, (xii) K2CO3, MeCN.
16766 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
Crystallography
(Fig. S1, ESI†). The resulting values were determined as τD O =
2
2.8 ms and τH O = 1.9 ms. Using the empirical relationship
2
The thus far unreported crystal structure of Quin C1 (6) was
acquired by slow evaporation from methanol at room tempera-
ture and is depicted in Fig. 3. The compound was found to
crystallise as a mixture of both the R and S enantiomer (chiral
carbon: C3), which stacked alternately. In the solid state, two
twists within the molecule along the C3–C24 bond and the
C12–C13 bond were observed, creating three planes. Bond
lengths and angles are listed in the ESI.† Intermolecular
H-bonding between the O1 and H4A (on N4) atoms was
observed with a bond distance calculated as 2.27 Å (Fig. 3B).
between the lifetimes of a species in H2O and D2O and the
number of bound water molecules respectively,44,45 the
hydration number (q-value) was determined as q = 0.8,
suggesting an octadentate ligand environment and a 9-coordi-
nate metal centre.
In order to assess the short-lived, ligand-based components
of the emission, Time-Correlated Single Photon counting
(TCSPC) studies were undertaken on a PicoQuant FluoTime
300 equipped with a 375 nm picosecond pulsed laser. The
resulting decay curves for short lifetimes were fitted using a
numerical reconvolution algorithm to account for the finite
Instrument Response Function (IRF) using FluoFit software.
Interestingly, the fluorescence decay obtained for Tb·14 by this
method could not be fitted to a single exponential decay curve,
but instead was best fitted to a biexponential decay with 2
components with ns range lifetimes of τ1 = 11 ns (approxi-
mately 85% fractional intensity) and τ2 = 3 ns (approximately
15% fractional intensity) (Fig. S2, ESI†). This phenomenon is
tentatively assigned to dual emission occurring from two
orthogonal components of the organic system, which is sup-
ported by the observation of two ligand-based emission pro-
cesses in the complex (see below). The preferred non-planar
conformation around the diacyl hydrazine linkage will prevent
efficient relaxation through dipole–dipole interactions and pre-
serve independent emission from each component (see Fig. 3).
In the europium complex (Eu·14), weak lanthanide emis-
sion was observed upon application of a time gate, while
steady state emission spectra were comparable to those of the
Photophysical properties
Steady-state and time-gated luminescence and absorption
spectra of Tb·14 were recorded in aerated methanol solutions
(Fig. 4A). Excitation at 350 nm yielded an emission maximum
at 441 nm. Two maxima at 227 nm and 347 nm (λem = 440 nm)
were apparent within the excitation spectrum. Upon appli-
cation of a 0.1 ms time gate and excitation at 350 nm, charac-
teristic terbium emission bands with maxima at 490 nm,
546 nm, 586 nm and 620 nm arising from 5D4
→
7FJ tran-
sitions, for J = 6, 5, 4 and 3 respectively were apparent within
the spectrum, demonstrating sensitisation of the lanthanide
via the ligand chromophore. Metal-centred luminescence life-
times of Tb·14 were recorded in water and deuterium oxide
and their decays were fitted to a single-exponential function
terbium analogue (λmax = 451 nm for emission and λmax
=
348 nm for the longest wavelength excitation band in metha-
nol, Fig. S3, ESI†). The faint lanthanide-based emission is
likely indicative of a poor energy match between the relevant
excited state within the antenna unit and the 5D0 europium
emissive state.
Typically, population of the lanthanide excited state is
known to proceed via an energy transfer process (sensitis-
ation).46 Often, this pathway occurs through ligand excitation,
intersystem crossing to the ligand-based excited triplet state
followed by an energy transfer to the relevant emissive lantha-
nide state.47 Gadolinium-centred emission occurs within the
UV range of the spectrum and thus does not interfere with the
visible light ligand-based emission, making the gadolinium
analogue (Gd·14) a convenient lanthanide complex model to
study ligand-based emission.48
To observe phosphorescence (T1 → S0) from the ligand
triplet energy level (T1) which is typically considered respon-
sible for sensitising the metal unit, emission spectra of Gd·14
under low temperature conditions were recorded (to minimise
triplet state deactivation by non-radiative processes). The
obtained spectra revealed that upon cooling to 77 K in
ethanol, an intense transition at 416 nm and a weak
maximum at 539 nm were apparent (Fig. 4B). The weak, lower
energy band was tentatively assigned to the ligand-centred
triplet state, whereas the shift to higher energy emission at low
Fig. 4 (A) Excitation spectrum (red), steady state emission spectrum
(yellow) and emission spectra with an applied time-gate of 0.1 ms (blue)
of compound Tb·14 in methanol (B) emission spectra of Gd·14 at room
temperature (dark blue) and 77 K (light blue) in ethanol.
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16767

View Article Online
Paper
Dalton Transactions
temperature (441 nm at room temperature to 416 nm at 77 K) in water. This value is surprisingly high, considering that low
is indicative of a charge transfer process.49 These results led us molecular weight, octadentate gadolinium chelates typically
to hypothesise that the ligand-based emission seen at room exhibit r1 values of 3–5 mM−1 s−1 39
which may be driven by
,
temperature could result from a thermally activated ligand the observed increase in TSAP isomer compared to the starting
charge transfer band (supported by the broad, structureless material. Although used as an indication, this value does not
nature of emission and absorption bands with large Stokes’ predict how well the compound would perform in a more com-
shifts), while a shift to higher energies of that band are plicated in vitro or in vivo setting.
observed at low temperature. The data further led us to hypoth-
esise that an unusual lanthanide sensitisation pathway in the
In vitro viability assessment
terbium complex may occur via the energetically well-matched
charge transfer band (at 22 575 cm−1 compared to the Tb(III)
For Tb·14 to provide a useful platform for imaging inflam-
mation via FPR2/ALX, it is crucial to ensure the compound
5D4 excited state at 20 400 cm−1),50 whereas the tentatively
assigned ligand triplet state (at 18 553 cm−1) appears too low
itself does not modify the normal immunological functions of
neutrophils (such as their ability to move towards a chemical
in energy for terbium sensitisation. Additionally, our results
gradient, transmigrate through an endothelial layer and their
suggest that neither the ligand triplet state nor the charge
granular content release e.g. myeloperoxidase [MPO]). With
this in mind, neutrophils were pre-treated with saline control
transfer state can efficiently sensitise europium emission
(Fig. S3, ESI†).
or varying, biologically relevant concentrations of the com-
Similar sensitisation pathways via an antenna charge trans-
pound (10−6, 10−7, 10−8 and 10−9 M) and neutrophil functional
fer excited state have been studied extensively for Eu(III), Yb(III)
and Nd(III) complexes,51,52 whereas examples of intra ligand
responses were analysed using three distinct assays to assess
charge transfer (ILCT)-sensitised Tb(III) emission are rare.49
chemotaxis, transmigration and MPO release (Fig. 6). As the
initial recruitment of neutrophils in inflammation is driven by
This sensitisation pathway, which proceeds via the strong,
their ability to move towards a chemical signal or chemoattrac-
ligand-based absorption band that is typically associated with
an ILCT state, could be harnessed for microscopy applications.
tant (e.g. leukotriene B4 [LTB4]), we tested the effect of neutro-
phils to migrate towards LTB4 (10−6 M) or PBS vehicle with and
Investigation into conformational equilibria
without pre-treatment with Tb·14 (Fig. 5). The data collected
was expressed as a migration index (ratio of neutrophils
migrated in response to LTB4 and neutrophils migrated in
response to vehicle) and statistical analysis confirmed that at
relevant compound concentrations, no significant change in
neutrophil mobility was observed compared to the PBS control
(Fig. 6A).
An important part of the inflammatory response is the
ability of neutrophils to extravasate from blood vessels to the
site of tissue injury.63 To simulate this biological function,
neutrophils were incubated with PBS control or varying con-
centrations of Tb·14 (10−6, 10−7, 10−8 and 10−9 M) and their
ability to migrate through a HUVEC layer towards a chemoat-
tractant (LTB4) was assessed. Fig. 6B shows that the compound
Ln(III)-DOTA complexes form two well characterised stereoi-
somers that are visualised as a square antiprism (SAP) isomer
and a twisted square antiprism (TSAP) isomer. The two
isomers can interconvert in solution and differ in the twist
angle between the oxygen and the nitrogen binding
planes.53,54 The equilibrium between the two species can be
tuned by different factors55–57 and is particularly relevant to
gadolinium complexes with uses in MRI, as the TSAP geometry
is often associated with substantially increased water exchange
rates and correspondingly higher relaxivity values.58,59
Within the 1H-NMR spectrum of Eu·14 (Fig. S4A, ESI†) reso-
nances consistent with the SAP axial proton region
(29–35 ppm) and the minor TSAP isomer axial protons
(between 10 and 14 ppm) were present.60 The signals appear
broadened compared to those observed in the starting material
(Fig. S4B, ESI†) and more than four proton environments were
observed, likely arising from further, superimposed confor-
mational equilibria.61 In addition, the spectrum suggests an
increase in the amount of the minor TSAP isomer compared to
the starting material (estimated prevalence by relative inte-
gration 0.36 : 1), consistent with previous reports that increas-
ing steric demands on the lanthanide ion leads to an
increased preference for the TSAP geometry.57
Relaxometric measurements
Fig. 5 Schematic of chemotaxis assay showing how freshly isolated
human neutrophils incubated with different concentrations of Tb·14
were added to the top wells of a Neuroprobe chemotaxis chamber,
whilst LTB4 (10−6 M) was added to the bottom wells to assess whether
the compound affects neutrophil chemotactic responses. Example
Next, the longitudinal relaxation time (T1) of Gd·14 in water
was determined at distinct molar concentrations (at 400 MHz
and 294 K) using inversion recovery experiments. The resulting
relaxivity values were estimated according to a graphical pro-
cedure62 and a value of 6.9 mM−1 s−1 was measured for Gd·14 images of migrated neutrophils are shown (scale bar = 100 μm).
16768 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
the ability of neutrophils to release their MPO, however,
we found that an attenuation of granular release occurred at
10−8 M, whereas the presence of certain Tb·14 concentrations
(10−7 M and 10−9 M) produced little MPO effects. These results
suggest that Tb·14 at a concentration of 10−6 M is more suit-
able as an imaging agent as this concentration does not
inhibit the ability of neutrophils to release their MPO i.e.
perform their immune response.
Proof of concept time-resolved microscopy investigation
Human neutrophils were isolated and pre-treated with vehicle
(PBS) or stimulated with TNFα. Neutrophils were subsequently
incubated with Tb·14, fixed and prepared for visualisation
using a time-resolved confocal fluorescence microscope. To
obtain terbium-based signals, emission was recorded as a life-
time map using pulsed 375 nm laser excitation.
At concentrations of 10−4 M to 10−6 M, no metal-based
signals were observed. Upon increasing probe concentrations
to 10−3 M, ring-like structures of pseudo-intensity on the cellu-
lar periphery were evident within the stimulated group,
(Fig. 7A), as were randomly speckled areas of increased signal.
Within the stimulated group, presence of terbium-based
signals were found, whereas no long-lived emission com-
ponents were obtained in unstimulated samples (Fig. 7B). The
overall averaged cellular decay times within the stimulated
group of ∼20 μs are short compared to lanthanide-only emis-
sion observed in a cuvette. However, it should be noted that
these preliminary data were obtained using TCSPC methods
Fig. 6 (A) Chemotaxis data represented as migration index (calculated
by dividing numbers of neutrophils migrated to LTB4 by numbers of
cells migrated to vehicle [phosphate buffered saline (PBS)]). (B)
Transmigration data expressed as the number of migrated neutrophils
through an endothelial cell layer (human umbilical vein endothelial cells
[HUVECs]) per unit volume in response to LTB4 in presence of varying
Tb·14 concentrations or absence of the compound. (C)) quantification
of neutrophil myeloperoxidase (MPO) release upon stimulation using
relative MPO activity compared to a standard. All data are mean SEM
of 3–4 independent donors run in duplicate. *p < 0.05 vs. corresponding
vehicle control group, #p < 0.05 vs. LTB4 control group (Friedman and
Dunn’s test).
did not modify the ability of neutrophils to transmigrate
towards LTB4 at any of the concentrations tested. Although
there appeared to be a trend towards less neutrophils
migrating towards LTB4 when treated with the lower Tb·14 con-
centrations (i.e. 10−7 to 10−9 M), this was not significant when
compared to the LTB4 control. Equally, all groups (i.e. the LTB4
control group and all Tb·14 concentrations tested) produced
significant effects when compared with the PBS control group,
authenticating the use of Tb·14 as an imaging agent.
Under inflammatory conditions neutrophils release reactive
species from their intracellular granules in order to eliminate
invading pathogens. Amongst these granular proteins, myelo-
peroxidase (MPO, a marker of neutrophil activation) is known
to perform anti-microbial functions as well as to contribute
significantly to neutrophil dependent inflammation.64 To test
Fig. 7 (A) Lifetime maps of neutrophils obtained using a time-resolved
confocal microscope to visualise uptake of Tb·14 in unstimulated cells
whether presence of Tb·14 changes MPO production in acti- or cells that had been treated with TNFα prior to incubation with the
compound. Long-lived signals around the cell periphery indicate pres-
ence of the compound, while no long-lived decay components were
detected in unstimulated samples. (B) Luminescence lifetime decay and
tail-fitting (shown in red) of the overall average decay in a stimulated
vated neutrophils compared to the control, we assessed MPO
activity in neutrophils that had transmigrated through an
endothelial (HUVEC) layer. In this assay, we found a concen-
tration dependent response of Tb·14 on neutrophil MPO pro-
neutrophil, demonstrating terbium-based emission and an average cell
duction. At high concentrations (10−6 M), Tb·14 did not affect lifetime of ∼20 μs.
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16769

View Article Online
Paper
Dalton Transactions
including the entire range of emission lifetimes, rather than spectra were recorded on a Bruker AMX-500 spectrometer at
using time-gated methods to measure phosphorescence alone. room temperature. Coupling constants are quoted in
Hence, the calculated average lifetimes in Fig. 7 include all cel- Hertz (Hz) and multiplicities are abbreviated as: s = singlet,
lular emission including autofluorescent background signals d = doublet, t = triplet, q = quartet, m = multiplet and br =
(typically sub 10 ns) and short-lived Quin C1-based emission. broad. Electrospray Time-of-Flight and MALDI mass spectra
Although lanthanide complexes are typically less prone to were obtained using a Waters LCT Premier. Thin-layer chrom-
quenching by cellular material than organic dyes due to the atography was conducted using pre-coated Silica gel 60, F254
triplet nature of the emission, it is also possible that the cellu- plates with a thickness of 0.2 nm. Column chromatography
lar lifetime of the complex differs from the solution lifetime. was performed using silica gel or Sephadex-G10 and laboratory
However, while the average lifetime method does not demon- grade solvents, under mild pressure.
strate any ms component to the decay, the observed 20 µs
average is a demonstration of uptake of the complex in stimu-
Synthetic procedures
lated cells being in the order of 103 × the average lifetime Compounds 2, 3, 4, 5 and 6 were prepared as detailed below,
observed in unstimulated cells. The pattern of attachment using procedures that had been modified from ref. 22.
observed in stimulated samples is consistent with images pre-
Methyl 4-butoxybenzoate (2)
viously observed using fluorescently tagged peptides.65 In the
study, attachment to the FPRs in unstimulated neutrophils Potassium carbonate (6.83 g, 50 mmol) and methyl 4-hydroxy-
occurred uniformly distributed throughout the cell. In con- benzoate (5.00 g, 33 mmol) were dissolved in methanol
trast, for stimulated neutrophils peptide-labelled receptors (60 mL) and stirred under nitrogen at 0 °C for 20 minutes.
accumulated around the cell periphery or, in a further cell 1-Bromobutane (4.97 g, 37 mmol) was added dropwise before
subset, in areas of receptor clusters.
the mixture was heated at reflux for 23 hours. The solvents
were removed in vacuo, the reaction mixture was dissolved in
dichloromethane (50 mL), washed with water (3 × 50 mL),
brine (2 × 50 ml), dried over magnesium sulphate and concen-
trated. Purification was achieved by silica column chromato-
Conclusions
Lanthanide frameworks tethered to a small-molecule FPR2/ graphy in dichloromethane/petroleum ether (3 : 1) and yielded
1
ALX ligand were described in this work and their ability to act a clear oil (4.09 g, 60%). H-NMR (400 MHz, CDCl3) δH (ppm):
3
3
as cellular imaging probes was investigated. The targeting 0.99 (3H, t, JHH = 7.4 Hz), 1.51 (2H, m, JHH = 7.6 Hz), 1.80
3
3
moiety, Quin C1, was found to act as an efficient sensitising (2H, m, JHH = 6.5 Hz), 3.89 (3H, s), 4.02 (2H, t, JHH = 6.5 Hz),
unit for terbium-emission, whereas the europium analogue 6.91 (2H, d, 3JHH = 8.9 Hz), 7.98 (2H, d, 3JHH = 8.9 Hz).
appeared largely non-luminescent under time-gated con-
ditions. Proton NMR spectroscopy of Eu·14 revealed typical
DOTA-type signals. Attachment of the targeting group was
CI+: m/z 209 {M + H}+, 226 {M + NH4}+.
4-Butoxybenzohydrazide (3)
found to result in a relative increase of the TSAP isomer (and Compound 2 (2.70 g, 13 mmol) and hydrazine monohydrate
thus preferable water exchange properties), which was also (2.60 g, 52 mmol) were dissolved in ethanol (50 mL) and
reflected in the comparatively high r1 value of 6.9 mM−1 s−1 at heated at 65 °C under a nitrogen atmosphere for 24 hours,
400 MHz and 294 K (compared to typical r1 values of before further 4 equivalents of hydrazine monohydrate were
∼3–5 mM−1 s−1 for commercially used contrast agents based added. The reaction was heated for further 30 hours.
on octadentate metal-binding frameworks). In vitro testing of Subsequently, the reaction mixture was concentrated in vacuo
compound Tb·14 was conducted to examine the effects on neu- and the resulting oil was purified by activated alumina column
trophil functional responses in physiological and pathological chromatography (Brockman grade V), eluting initially with di-
conditions. We ascertained that neutrophil functions chloromethane. Once the starting material had eluted, a
remained intact at certain concentrations of Tb·14. Despite methanol gradient (up to 20%) was added to afford a crystal-
weak cellular signals, a time-resolved microscopy proof of line white solid (1.90 g, 70%). 1H-NMR (400 MHz, CDCl3) δH
3
3
concept experiment confirmed preferential uptake of Tb·14 (ppm): 0.99 (3H, t, JHH = 7.4 Hz), 1.51 (2H, m, JHH = 7.6 Hz),
3
3
occurred in activated cells, and distinctive cellular mor- 1.79 (2H, m, JHH = 6.5 Hz), 4.01 (2H, t, JHH = 6.5 Hz), 4.07
3
phologies were observed, whereas no observable long-lived (2H, br, –NH2
̲
), 6.93 (2H, d, JHH = 8.9 Hz, Ha), 7.70 (2H, d,
signals were noted in unstimulated groups.
3JHH = 8.9 Hz, Hb). ES+: m/z 209 {M + H}+.
2-(4-Butoxybenzoyl)hydrazide 2-nitro-benzoic acid (4)
4-Butoxybenzohydrazine (1.80 g, 9 mmol) and potassium car-
bonate (1.20 g, 9 mmol) were dissolved in dichloromethane at
0 °C under a nitrogen atmosphere. 2-Nitro-benzoyl chloride
Experimental
Materials and general procedures
Reagents were bought from Sigma Aldrich or Fisher Scientific (1.0 mL, 7.8 mmol) was added slowly to the solution, which
and used without further purification. Deuterated solvents was gradually allowed to warm to room temperature. After stir-
were bought from Goss Scientific. 1H-NMR and 13C-NMR ring for further 15 hours, the reaction mixture was washed
16770 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
with water (2 × 50 mL) and brine (2 × 50 mL), the organic frac- (2 × 5 mL) and dried over magnesium sulphate. Purification
tions were combined and dried over magnesium sulphate was achieved by silica column chromatography eluting with a
before the remaining solvents were removed in vacuo to yield a dichloromethane/acetone mixture (95 : 5) to afford an off-white
1
pale yellow solid (1.38 g, 43%). 1H-NMR (400 MHz, MeOD) solid (0.07 g, 60%). H-NMR (400 MHz, d6-acetone) δH (ppm):
3
3
3
3
δH (ppm): 1.01 (3H, t, JHH = 7.4 Hz), 1.53 (2H, m, JHH
=
0.97 (3H, t, JHH = 7.4 Hz), 1.50 (2H, m, JHH = 7.6 Hz), 1.78
7.4 Hz), 1.80 (2H, m, 3JHH = 6.4 Hz), 4.07 (2H, t, 3JHH = 6.4 Hz), (2H, m, JHH = 7.2 Hz), 3.72 (3H, s), 4.08 (2H, t, JHH = 6.5 Hz),
3
3
7.01 (2H, d, 3JHH = 8.9), 7.72–8.16 (6H, br).
ES+: m/z 358 {M + H}+.
4.84 (2H, br), 6.81 (2H, dt, 3Jortho = 8.9 Hz, 4Jmeta = 2.1 Hz), 7.02
(2H, m), 7.13 (1H, br, s), 7.39 (3H, m), 7.58 (2H, m), 7.96 (3H,
m), 10.37 (1H, br). 13C-NMR (400 MHz, d6-acetone) δH (ppm):
13.1, 18.8, 30.9, 54.5, 67.6, 113.7, 114.1, 124.0, 126.4, 127.7,
2-(4-Butoxybenzoyl)hydrazide 2-amino-benzoic acid (5)
Compound 4 (1.38 g, 3.8 mmol) was dissolved in a mixture of 128.5, 129.6, 132.9, 159.8, 160.4, 162.5, 165.7.
acetic acid and dichloromethane (1 : 1, 80 mL) at 0 °C. Excess
HRMS: m/z calculated: 552.1790, found: 552.1793 for
zinc dust (2.50 g, 38.4 mmol) was added to the stirring {M + H}+.
mixture which was subsequently gradually allowed to warm to
N-(tert-Butoxycarbonyl)-1,2-diaminoethane (9),40 N-(tert-
room temperature. After 16 hours, the mixture was filtered butoxycarbonyl)-N′-aminoacetylchloride (10),41 1,4,7-tris(tert-
through Celite and washed with dichloromethane (10 × butoxycarbonylmethyl)-10-(N-(2-tertbutoxycarbonylaminoethyl)
20 mL). The solvents were removed in vacuo, before the reac- acetamide)-1,4,7,10-tetraazacyclododecane (11)42 and 1,4,7-tris
tants were dissolved in dichloromethane, washed with satu- (carbonylmethyl)-10-(aminoethyl-N-acetyl)-1,4,7,10-tetraazacy-
rated sodium bicarbonate (3 × 50 mL) and dried over mag- clododecane (12)42 were prepared as detailed in the respective
nesium sulphate to yield an off-white solid (0.87 g, 70%). references.
3
1H-NMR (400 MHz, MeOD) δH (ppm): 1.01 (3H, t, JHH
=
3
3
Preparation of Ln·13 42
7.4 Hz), 1.54 (2H, m, JHH = 7.6 Hz), 1.79 (2H, m, JHH = 6.4
Hz), 4.07 (2H, t, 3JHH = 6.4 Hz), 6.75 (2H, m), 7.02 (2H, m), 7.32 Compound 12 and the respective lanthanide salt were dis-
(1H, m), 7.73 (1H, m), 7.90 (2H, m). ES+: m/z 368 {M + solved in water in a 1 : 1.1 molar ratio, the pH was adjusted to
MeCN}+.
5.5 and the solution was stirred overnight at room tempera-
ture. Subsequently, the pH of the mixture was adjusted to 10
using a sodium hydroxide solution (1 M) and stirred for
Quin C1 (6)
2-(4-Butoxybenzoyl)hydrazide 2-amino-benzoic acid (0.54 g, 45 minutes. The suspension was centrifuged and filtered to
1.6 mmol) and 4-methoxy benzaldehyde (0.27 g, 2.0 mmol) remove inorganic solids. Next, the pH of the filtrate was read-
were dissolved in ethanol (20 ml) and heated at reflux for an justed to 7 using a hydrochloric acid solution (1 M) and a
hour before addition of citric acid (0.38 g, 2.0 mmol). The xylenol orange test was performed. Water was removed
reagents were further refluxed for 48 hours. Residual solvent in vacuo and the residue was recrystallised from a methanol
was removed in vacuo and the reaction mixture was dissolved and diethyl ether mixture and repeatedly washed with diethyl
in dichloromethane and filtered. The filtrate was purified by ether. Tb·13: (0.05 g, 59%). IR (solid) in cm−1: 3472, 2359,
silica column chromatography eluting with a dichloromethane 1599, 1438, 1406, 1256, 1168, 1036. HRMS: m/z calculated:
and acetone gradient of 0%–50% to yield a white solid (0.59 g, 603.1575 found: 603.1580 for {M + H}+. Eu·13: (0.21 g, 88%).
83%). 1H-NMR (400 MHz, d6-acetone) δH (ppm): 0.95 (3H, t, 1H-NMR (400 MHz, D2O) δH (ppm): −16.66, −16.20, −15.60,
3JHH = 7.4 Hz), 1.47 (2H, m, 3JHH = 7.6 Hz), 1.74 (2H, m, 3JHH
=
−14.97, 14.67, −14.19, −13.91, −12.60, −11.58, −10.91, −9.41,
3
6.5 Hz), 3.78 (3H, s), 4.02 (2H, t, JHH = 6.5 Hz), 6.30 (2H, br), −8.47 to −6.95, −6.68, −5.53, −5.02, −4.25, −3.24, −2.31,
6.83 (1H, td, 3Jortho = 7.1 Hz, 4Jmeta = 1.0 Hz), 6.90 (5H, m), 7.35 −1.72, −1.03, −0.45, 0.38, 1.17, 1.48, 2.49, 2.81–4.51, 6.78,
3
4
3
(1H, td, Jortho = 8.0 Hz, Jmeta = 1.6 Hz), 7.49 (2H, d, Jortho
=
10.53, 11.30, 11.77–12.85, 13.52, 30.74, 31.88, 33.70, 34.07.
8.6), 7.66 (2H, m), 7.84 (1H, dd, 3Jortho = 7.8 Hz, 4Jmeta = 1.5 Hz), HRMS: m/z calculated: 595.1531 found: 595.1548 for {M + H}+.
9.38 (1H, br). 13C-NMR (400 MHz, d6-acetone) δH (ppm): 14.1, Gd·13: (0.05 g, 35%). HRMS: m/z calculated: 602.1574, found:
19.8, 31.9, 55.6, 68.5, 74.8, 114.4, 114.8, 115.5, 119.1, 129.3, 602.1576 for {M + H}+.
130.2, 130.4, 134.6, 149.7, 161.4, 162.9, 165.3, 166.3, 180.7.
Preparation of Ln·14
HRMS: m/z calculated: 446.2080, found: 446.2089 for
{M + H}+.
Potassium carbonate and Ln·13 (in 5 : 1 molar ratios) were sus-
pended in acetonitrile and stirred for half an hour before
addition of 7 (1 equivalent). The mixture was heated at 60 °C
Quin C1-acetyl chloride (7)
Compound 6 (0.10 g, 0.22 mmol) and triethylamine (0.03 g, for two days. Inorganic salts were removed by filtration and
0.33 mmol) were dissolved in dichloromethane (3 mL) under a residual solvents were evaporated to yield a white solid. The
nitrogen atmosphere. The stirring solution was immersed in crude product was isolated by Sephadex G-10 size exclusion
an ice/acetone bath and chloroacetyl chloride (0.030 g, chromatography eluting with MilliQ water. Tb·14: (0.02 g,
0.26 mmol) in dichloromethane (2 mL) was added dropwise. 25%). IR (solid) in cm−1: 2361, 1607, 1251, 1173. HRMS: m/z
The reaction mixture was gradually warmed to room tempera- calculated: 1089.4792, found: 1089.4794 for {M + H}+. Eu·14:
ture and stirred overnight, before it was washed with water 1H-NMR (400 MHz, D2O) δH (ppm): −17.17 to −15.97, −15.76,
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16771

View Article Online
Paper
Dalton Transactions
−13.40, −12.79 to −10.07, −9.37, −8.25 to −6.79, −6.59, −5.38, Relaxometric measurements
−4.85, −3.28, −2.59 to −1.77, −1.00, −0.45, 0.51, 0.96, 1.26,
Gd(III) complexes were dissolved in water at 3 different molar
concentrations. The three solutions and a water standard were
added into a glass capillary of 1.7 mm diameter, sealed and
inserted into an NMR tube containing D2O. Concentrations of
Gd(III) present were assessed by measuring the difference in
solvent shifts according to the magnetic susceptibility
measurements conducted using the Evans method.69 T1
measurements were carried out using a Bruker Advance 400
spectrometer and inversion recovery measurements. Relaxivity
values were calculated from the samples using a graphical pro-
cedure as described in the literature.62 The longitudinal relax-
ation time (T1) of Gd·14 in water was determined at three dis-
tinct molar concentrations on a 400 MHz NMR spectrometer
at 294 K. In addition, T1 for just water (T1,0) was determined
under equivalent conditions.
1.48, 1.79, 1.91, 2.09, 2.69, 3.52–4.27, 5.89, 5.94, 5.99,
6.72–8.55, 10.92–14.33, 29.55–34.30. HRMS: m/z found:
1182.5885 for {M + H2O + 2 MeCN + H}+. Gd·14: (0.02 g, 30%).
HRMS m/z: calculated: 1087.3524, found: 1087.3661 for {M + H}+.
X-ray crystal structure of 6
Crystal data for 6: C26H27N3O4, M = 445.50, monoclinic, P21/c
(no. 14), a = 14.1692(8), b = 13.5603(6), c = 12.7578(7) Å, β =
110.407(7)°, V = 2297.4(2) Å3, Z = 4, Dc = 1.288 g cm−3, μ(Mo-
Kα) = 0.088 mm−1, T = 173 K, colourless tabular needles,
Agilent Xcalibur
measured reflections (Rint
3
E
diffractometer; 4582 independent
=
0.0174), F2 refinement,66,67
R1(obs) = 0.0527, wR2(all) = 0.1405, 3195 independent observed
absorption-corrected reflections [|Fo| > 4σ(|Fo|), completeness
to θfull(25.2°) = 98.8%], 316 parameters. CCDC 1945848.†
The C3-bound p-methoxyphenyl group, as well as C3 itself,
in the structure of 6 was found to be disordered. Two orien-
tations were identified of ca. 73 and 27% occupancy, their geo-
metries were optimised, the thermal parameters of adjacent
atoms were restrained to be similar, and only the non-hydro-
gen atoms of the major occupancy orientation were refined
anisotropically (those of the minor occupancy orientation were
refined isotropically). As a consequence of this disorder the
N4–H hydrogen atom was also disordered across two sites, and
so rather than being found from a ΔF map the two orien-
tations were added in idealised positions with riding thermal
parameters. The N11–H hydrogen atom, by contrast, was
located from a ΔF map and refined freely subject to an N–H
distance constraint of 0.90 Å.
Cellular studies
All in vitro studies were performed at LSU Health Sciences
Center, Shreveport.
Neutrophil isolation
Blood samples were collected by healthcare professionals from
healthy volunteers after obtaining informed consent (50 mL).
The study was approved by the institutional review board of
the LSUHSC-S (STUDY00000261) and conducted in accordance
with the Declaration of Helsinki. After discarding approxi-
mately the first 3 mL (to avoid thrombin contamination),
45 mL of the sample were mixed with anticoagulant citrate
dextrose (ACD) solution (5 mL). The samples were centrifuged
(800 rpm for 20 minutes at room temperature) and the upper
layer of plasma was removed before adding 1× PBS (10 mL).
Subsequently, 6% dextran (v/w, 1.8 g in 30 mL 1× PBS) solution
(8 mL) was added, the sample was gently inverted several
times and left to sediment for 15 minutes. The leukocyte layer
was collected and separated over a Histopaque 1077 gradient
(10 mL per tube). After centrifugation (1500 rpm for
30 minutes at room temperature), the supernatant was
removed. Cold water (9 mL) was added in order to lyse residual
red blood cell, and subsequently mixed with 10× PBS (1 mL).
The volume was made up to 40 mL using 1× PBS and centri-
fuged (1000 rpm for 10 minutes at room temperature). The
supernatant was removed, and the cells were resuspended in
1× PBS (5 mL) and the procedure was repeated. Finally, neutro-
phils were resuspended DMEM with 3% FCS (10 mL). The cells
were counted using a Neubauer hemocytometer (diluted 1 : 1
with trypan blue) and the resulting cell count was diluted
respectively for the number of cells required per assay
performed.
Photophysical measurements
UV-vis absorption spectra were recorded on a PerkinElmer 650
spectrometer in quartz cuvettes. Concentrations were adapted
to ensure a sensible absorbance range. Fluorescence excitation
and emission spectra were obtained using a Varian Cary
Eclipse spectrophotometer.
Lanthanide-based lifetimes were measured using a Varian
Cary Eclipse spectrophotometer in the phosphorescence
mode, using 1 mM solutions in water or deuterium oxide. The
integrated intensity was recorded after excitation of the
samples at 350 nm. The gate time was fixed at 0.1 ms with
both excitation and emission slits set at 20 nm. The resulting
decay curves were fitted to a single exponential decay in
Microsoft Excel. Hydration number (q-values) were calculated
according to the following equation.45
ꢀ1
ꢀ1
q ¼ AðτH O ꢀ τD O ꢀ BÞ
2
2
with A = 5 ms and B = 0.06 ms−1 for terbium.68
Chemotaxis assay
Short-lived organic component luminescence lifetimes were
assessed in methanol solutions on a time resolved PicoQuant Chemotaxis assays were run using 3 μm pore size ChemoTx®
FluoTime 300 with a 375 nm picosecond pulsed laser using System 96 well plates (Neuro Probe, USA). LTB4 (10−6 M, 29 μL,
TCSPC. Fitting of the luminescence decay was conducted in Sigma Aldrich) as a chemotactic stimulus or PBS as a control
FluoFit software.
(29 μL) were added to the bottom of the wells. Freshly isolated
16772 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
neutrophils at a concentration of 4 × 106 cells per mL in
DMEM and 3% fetal calf serum (FCS) were treated with vehicle
Conflicts of interest
(PBS) or varying concentrations of the compound for There are no conflicts to declare.
10 minutes prior to addition to the top of the well plate mem-
branes (25 μL). The plates were incubated for 3 hours at 37 °C
with 5% CO2 before removal of the membrane. Migrated cells
were manually counted using a Neubauer hemocytometer after
Acknowledgements
dilution in a 1 : 1 ratio with trypan blue.
This work was supported by the UK Engineering and Physical
Sciences Research Council (EPRSC) grant number EP/L016737/
1. Dr F. N. E. Gavins acknowledges the support of the Royal
Society Wolfson Fellowship under grant number RSWF/R3/
183001. N. J. L. is grateful for a Royal Society Wolfson Research
Merit Award.
Transmigration assay
To assess cellular transmigration, 24-well plate inserts were
treated with fibronectin for 30 minutes, before a monolayer of
human umbilical vein endothelial cells (HUVECs, 32 000 cell
per 200 μL) were grown for 72 hours. HUVEC media was
removed and neutrophils (106 cells per mL in DMEM and 3%
FCS) that had been pre-treated with vehicle or varying com-
pound concentrations for 10 minutes, were added to the
inserts (500 μL). PBS (vehicle, 500 μL) or LTB4 (10−6 M, 500 μL)
were added into a fresh 24-well plate set up and the inserts
were transferred before incubation (3 hours at 37 °C with 5%
CO2). Migrated cells were manually counted as described above.
Notes and references
1 P. Iribarren, J. Hu, Y. Le and J. M. Wang, Immunol. Res.,
2005, 1, 165–176.
2 R. Jin, G. Yang and G. Li, J. Leukocyte Biol., 2010, 87, 779–
789.
3 D. C. Rees, T. N. Williams and M. T. Gladwin, Lancet, 2010,
376, 2018–2031.
Myeloperoxidase assay
4 S. I. Grivennikov, F. R. Greten and M. Karin, Cell, 2010,
140, 883–899.
Myeloperoxidase (MPO) release was quantified from cells that
had been pre-activated by the transmigration assay. In order to
achieve this, the neutrophil supernatant (20 μL) was collected
from the bottom of transmigration plates and mixed in a 4 : 1
ratio with a 9 : 1 : 0.1 mixture of solutions A, B and C respect-
ively that had been previously prepared. Solution A was made
up of 5.4 g KH2PO4, 1.875 g EDTA and 5 mL Triton X-100.
Solution B was a solution of 3,3′,5,5′-tetramethylbenzidine
(TMB, 10 mM) in acetone (2 mL) and solution C was an
aqueous solution of hydrogen peroxide (30%, 25 µL) in water
(10 mL). The cellular mixture was incubated at room tempera-
ture for approximately 5 minutes until a colour change
occurred and the reaction was terminated by addition H2SO4
(1 M, 25 µL). Absorbance was read at 450 nm relative to a
standard.
5 F. N. E. Gavins, Trends Pharmacol. Sci., 2010, 31, 266–276.
6 C. N. Serhan and P. M. Murphy, Pharmacol. Rev., 2009, 61,
119–161.
7 J. G. Filep, M. Sekheri and D. El, Eur. J. Pharmacol., 2018,
833, 339–348.
8 Y. Le, P. M. Murphy and J. M. Wang, Trends Immunol.,
2002, 23, 541–548.
9 C. Dahlgren, M. Gabl, A. Holdfeldt, M. Winther and
H. Forsman, Biochem. Pharmacol., 2016, 114, 22–39.
10 S. A. Vital, F. Becker, P. M. Holloway, J. Russell, M. Perretti,
D. N. Granger and F. N. E. Gavins, Circulation, 2016, 133,
2169–2179.
11 D. A. Dorward, C. D. Lucas, G. B. Chapman, C. Haslett,
K. Dhaliwal and A. G. Rossi, Am. J. Pathol., 2015, 185, 1172–
1184.
12 G. J. Stasiuk, H. Smith, M. Wylezinska-Arridge,
J. L. Tremoleda, W. Trigg, S. K. Luthra, V. Morrison Iveson,
F. N. E. Gavins and N. J. Long, Chem. Commun., 2013, 49,
564–566.
Time-resolved microscopy
Neutrophils were pre-treated with TNFα (50 ng mL−1
30 minutes) or with PBS (control) and incubated with varying
,
concentrations of compound or vehicle for 45 minutes (37 °C, 13 L. W. Locke, M. D. Chordia, Y. Zhang, B. Kundu,
5% CO2). Cells were seeded on poly-L-lysine sheets (300 000
cells per sheet) and fixed using paraformaldehyde
(20 minutes) followed by direct transfer onto Fluoromont G
D. Kennedy, J. Landseadel, L. Xiao, K. D. Fairchild,
S. S. Berr, J. Linden and D. Pan, J. Nucl. Med., 2009, 50,
790–798.
coated glass slides. The sheets were firmly adhered to the 14 L. Xiao, Y. Zhang, Z. Liu, M. Yang, L. Pu and D. Pan, Bioorg.
slides using a nail base coat. Visualisation was performed on a Med. Chem. Lett., 2010, 20, 3515–3517.
MicroTime 200 (PicoQuant) time resolved confocal fluo- 15 Y. Zhang, L. Xiao, M. D. Chordia, L. W. Locke,
rescence microscopy system consisting of an Olympus IX73
confocal microscope fitted with a ×100 oil objective excited
M. B. Williams and S. S. Berr, Bioconjugate Chem., 2010, 21,
1788–1793.
with a picosecond pulsed 375 nm laser and coupled to a 16 G. J. Stasiuk, P. M. Holloway, C. Rivas, W. Trigg,
TimeHarp SPAD TCSPC unit. The single pixel decay profiles
were analysed using the SymphoTime 64 software package.
S. K. Luthra, M. Iveson, N. E. Gavins and N. J. Long, Dalton
Trans., 2015, 44, 4986–4993.
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16773

View Article Online
Paper
Dalton Transactions
17 J. Pellico, A. V. Lechuga-vieco, E. Almarza, A. Hidalgo, 40 W. Jiang, Q. Fu, H. Fan and W. Wang, Chem. Commun.,
C. Mesa, I. Fernández-Barahona, J. A. Quintana, J. Bueren, 2007, 259–261.
J. A. Enriquez, J. Ruiz-Cabello and F. Herranz, Sci. Rep., 41 A. Fanning, S. E. Plush and T. Gunnlaugsson, Chem.
2017, 7, 13242. Commun., 2006, 3791–3793.
18 J. W. Babich, R. G. Tompkins, W. Graham, S. A. Barrow and 42 W. S. Perry, S. J. A. Pope, B. J. Coe, M. Kenwright and
A. J. Fischman, J. Nucl. Med., 1997, 38, 1316–1320. S. Faulkner, Dalton Trans., 2010, 39, 10974–10983.
19 K. A. Stephenson, J. Zubieta, S. R. Banerjee, M. K. Levadala, 43 A. D. Sherry, P. Caravan and R. E. Lenkinski, J. Magn.
L. Taggart, L. Ryan, N. McFarlane, D. R. Boreham, Reson. Imaging, 2009, 1248, 1240–1248.
K. P. Maresca, J. W. Babich and J. F. Valliant, Bioconjugate 44 W. D. J. Horrocks and D. R. Sudnick, Acc. Chem. Res., 1981,
Chem., 2004, 15, 128–136. 14, 384–392.
20 K. A. Stephenson, S. R. Banerjee, T. Besanger, 45 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner,
O. O. Sogbein, M. K. Levadala, N. Mcfarlane, J. A. Lemon,
D. R. Boreham, K. P. Maresca, J. D. Brennan, J. W. Babich,
J. Zubieta and J. F. Valliant, J. Am. Chem. Soc., 2004, 126,
8598–8599.
D. Parker, L. Royle, A. S. de Sousa, J. A. G. Williams and
M. Woods, J. Chem. Soc., Perkin Trans. 2, 1999, 2, 493–
504.
46 J.-C. G. Bünzli, Coord. Chem. Rev., 2015, 293–294, 19–47.
21 T. M. Stepniewski and S. Filipek, Bioorg. Med. Chem., 2015, 47 J.-C. G. Bünzli and S. V. Eliseeva, Springer Ser. Fluoresc.,
23, 4072–4081. 2011, 13, 311–337.
22 M. Nanamori, X. Cheng, J. Mei, H. Sang, Y. Xuan, C. Zhou, 48 M. Shi, F. Li, T. Yi, D. Zhang, H. Hu and C. Huang,
M. Wang and R. D. Ye, Mol. Pharmacol., 2004, 66, 1213–
1222.
23 M. He, N. Cheng, W. Gao, M. Zhang, Y. Zhang, R. D. Ye
and M. Wang, Acta Pharmacol. Sin., 2011, 32, 601–610.
24 N. J. Long and W. T. Wong, The Chemistry of Molecular
Imaging, Wiley, 2015.
25 H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and
Y. Urano, Chem. Rev., 2010, 110, 2620–2640.
26 N. C. Shaner, P. A. Steinbach and R. Y. Tsien, Nat. Methods,
2005, 2, 905–909.
27 S. X. Fang and P. R. Chen, Chem. Commun., 2014, 50,
14724–14727.
Inorg. Chem., 2005, 44, 8929–8936.
49 A. D. Aléo, F. Pointillart, L. Ouahab, C. Andraud and
O. Maury, Coord. Chem. Rev., 2012, 256, 1604–1620.
50 A. J. Amoroso and S. J. A. Pope, Chem. Soc. Rev., 2015, 44,
4723–4742.
51 W. Lo, W. Wong and G. Law, RSC Adv., 2016, 6, 74100–
74109.
52 Z. Zhang, Y. Zhou, H. Li, T. Gao and P. Yan, Dalton Trans.,
2019, 48, 4026–4034.
53 M. Meyer, V. Dahaoui-Gindrey, C. Lecomte and R. Guilard,
Coord. Chem. Rev., 1998, 178–180, 1313–1405.
54 S. Aime, M. Botta, M. Fasano, M. P. M. Marques,
C. F. G. C. Geraldes, D. Pubanz and A. E. Merbach, Inorg.
Chem., 1997, 36, 2059–2068.
28 W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf
and C. Biskup, Microsc. Res. Tech., 2004, 63, 58–66.
29 M. Rajendran and L. W. Miller, Biophys. J., 2015, 109, 240– 55 I. J. F. Desreux, Inorg. Chem., 1980, 19, 1319–1324.
248.
56 S. Aime, M. Botta and G. Ermondi, Inorg. Chem., 1992, 19,
4291–4299.
30 W. Wang, K. Vellaisamy, G. Li, C. Wu, C.-N. Ko,
C.-H. Leung and D.-L. Ma, Anal. Chem., 2017, 89, 11679– 57 G. Tircso, B. C. Webber, B. E. Kucera, V. G. Young and
11684. M. Woods, Inorg. Chem., 2011, 50, 7966–7979.
31 C. Liu, C. Yang, L. Lu, W. Wang, W. Tan, C.-H. Leung and 58 A. C. L. Opina, M. Strickland, Y.-S. Lee, N. Tjandra,
D.-L. Ma, Chem. Commun., 2017, 53, 2822–2825.
32 Z. Mao, M. Wang, J. Liu, L.-J. Liu, S. M.-Y. Lee, C.-H. Leung
and D.-L. Ma, Chem. Commun., 2016, 52, 4450–4453.
33 J. Liu, C. Yang, C.-N. Ko, V. Kasipandi, B. Yang, M.-Y. Lee,
C.-H. Leung and D.-L. Ma, Sens. Actuators, B, 2017, 243,
971–976.
34 A. J. Amoroso and S. J. A. Pope, Chem. Soc. Rev., 2015, 44,
4723–4742.
35 C. P. Montgomery, B. S. Murray, E. J. New, R. Pal and
D. Parker, Acc. Chem. Res., 2009, 42, 925–937.
36 M. Pan, W. Liao, S. Yin, S. Sun and C. Su, Chem. Rev., 2018,
118, 8889–8935.
R. A. Byrd, R. E. Swenson and O. Vasalatiy, Dalton Trans.,
2016, 45, 4673–4687.
59 L. Dai, C. M. Jones, W. T. K. Chan, T. A. Pham, X. Ling,
E. M. Gale, N. J. Rotile, W. C. S. Tai, C. J. Anderson,
P. Caravan and G. L. Law, Nat. Commun., 2018, 9, 1–10.
60 D. Delli Castelli, M. C. Caligara, M. Botta, E. Terreno and
S. Aime, Inorg. Chem., 2013, 52, 7130–7138.
61 J. Blahut, P. Hermann, Z. Tošner and C. Platas-Iglesias,
Phys. Chem. Chem. Phys., 2017, 19, 26662–26671.
62 M. Rohrer, H. Bauer, J. Mintorovitch, M. Requardt and
H. Weinmann, Invest. Radiol., 2005, 40, 715–724.
63 K. Ley, C. Laudanna, M. I. Cybulsky and S. Nourshargh,
Nat. Rev. Immunol., 2007, 7, 678–689.
37 M. Pan, B. Du, Y. Zhu, M. Yue, Z. Wei and C.-Y. Su, Chem. –
Eur. J., 2016, 22, 2440–2451.
64 Y. Aratani, Arch. Biochem. Biophys., 2018, 640, 47–52.
38 K. Vellaisamy, G. Li, W. Wang, C. Leung and D. Ma, Chem. 65 V. M. Loitto, B. Rasmusson and K. E. Magnusson,
Sci., 2018, 9, 8171–8177. J. Leukocyte Biol., 2001, 69, 762–771.
39 C. Yang and K. Chuang, Med. Chem. Commun., 2012, 3, 66 G. M. Sheldrick, SHELXTL, version 5, Bruker AXS Inc.,
552–565.
Madison, Wisconsin, USA.
16774 | Dalton Trans., 2019, 48, 16764–16775
This journal is © The Royal Society of Chemistry 2019

View Article Online
Dalton Transactions
Paper
67 G. M. Sheldrick, Acta Crystallogr., Sect. C: Struct. Chem.,
2015, 71, 3–8.
M. Woods, J. Chem. Soc., Perkin Trans. 2, 1999, 2, 493–
504.
68 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, 69 D. M. Corsi, C. Platas-Iglesias, H. Van Bekkum and
D. Parker, L. Royle, A. S. de Sousa, J. A. G. Williams and
J. A. Peters, Magn. Reson. Chem., 2001, 39, 723–726.
This journal is © The Royal Society of Chemistry 2019
Dalton Trans., 2019, 48, 16764–16775 | 16775