Imidazole fused phenanthroline (PIP) ligands for the preparation of multimodal Re(i) and 99mTc(i) probes

Article abstract of DOI:10.1039/d0dt02829k

A small library of [2 + 1] 99mTc(i) complexes based on phenyl-imidazole-fused phenanthroline (PIP) ligands were synthesized and evaluated as multimodal molecular imaging probes. Using either a two-step or a one-pot synthesis method, 99mTc-PIP complexes containing N-methylimidazole as the monodentate ligand were prepared and isolated in good (54 to 89%) radiochemical yield, with the exception of one derivative bearing a strongly electron-withdrawing substituent. The stability of the [2 + 1] complexes was assessed in saline and in cysteine and histidine challenge studies, showing 6 hours stability, making them suitable for in vivo studies. In parallel, the Re(i) analogues were prepared as reference standards to verify the structure of the 99mTc complexes. The optical properties were consistent with other previously reported [2 + 1] type Re(i) complexes that have been used as cellular dyes and sensors. To facilitate the development of targeted derivatives, a tetrazine-PIP ligand was also synthesized. The 99mTc complex of the tetrazine PIP ligand effectively coupled to compounds containing a trans-cyclooctene (TCO) group including a TCO-albumin derivative, which was prepared as a model targeting molecule. An added benefit of the Re-PIP-Tz construct is that the emission from the metal complex was quenched by the presence of the tetrazine. Following the addition of TCO, there was a 70-fold increase in fluorescence emission, which can in future be leveraged during in vitro studies to reduce background signal. This journal is

Full text of DOI:10.1039/d0dt02829k

Dalton
Transactions
View Article Online
View Journal
PAPER
Imidazole fused phenanthroline (PIP) ligands for
the preparation of multimodal Re(I) and 99mTc(I)
probes†
Cite this: DOI: 10.1039/d0dt02829k
Samantha R. Slikboer,‡ Tamil Selvi Pitchumony,‡ Laura Banevicius, Natalie Mercanti,
Patricia E. Edem
and John F. Valliant
*
A small library of [2 + 1] 99mTc(I) complexes based on phenyl-imidazole-fused phenanthroline (PIP)
ligands were synthesized and evaluated as multimodal molecular imaging probes. Using either a two-step
or a one-pot synthesis method, 99mTc-PIP complexes containing N-methylimidazole as the monodentate
ligand were prepared and isolated in good (54 to 89%) radiochemical yield, with the exception of one
derivative bearing a strongly electron-withdrawing substituent. The stability of the [2 + 1] complexes was
assessed in saline and in cysteine and histidine challenge studies, showing 6 hours stability, making them
suitable for in vivo studies. In parallel, the Re(I) analogues were prepared as reference standards to verify
the structure of the 99mTc complexes. The optical properties were consistent with other previously
reported [2 + 1] type Re(I) complexes that have been used as cellular dyes and sensors. To facilitate the
development of targeted derivatives, a tetrazine-PIP ligand was also synthesized. The 99mTc complex of
the tetrazine PIP ligand effectively coupled to compounds containing a trans-cyclooctene (TCO) group
including a TCO-albumin derivative, which was prepared as a model targeting molecule. An added
benefit of the Re-PIP-Tz construct is that the emission from the metal complex was quenched by the
presence of the tetrazine. Following the addition of TCO, there was a 70-fold increase in fluorescence
emission, which can in future be leveraged during in vitro studies to reduce background signal.
Received 12th August 2020,
Accepted 29th September 2020
DOI: 10.1039/d0dt02829k
rsc.li/dalton
the probe is used to assist with margin determination during
surgical resection.6,12–16
Introduction
Multifunctional molecular imaging (MI) probes offer the
The typical approach used to prepare multimodal optical-
means to noninvasively monitor biochemical targets and pro- nuclear MI probes is to derivatize a targeting molecule with a
cesses in vitro and in vivo through the application of more fluorescent group and a radionuclide. The limitation here is
than one imaging modality.1–5 For example, molecules con- that the addition of two prosthetic groups can have a
taining both a radiolabeled prosthetic group and a near infra- detrimental influence on pharmacokinetics and the ability of
red (IR) fluorophore make it possible to employ nuclear the targeting molecule to retain affinity for the target. This is
imaging methods to assess whole-body biodistribution of a particularly true for small molecules. An alternative strategy
specific target while fluorescence microscopy can be used to is to use radiolabeled dyes5,6,17–26 or transition metal
visualize the expression of the target and distribution of the complexes9,11,27 that are luminescent and that have radioactive
probe within different cell lines and tissues.6–11 As well, single isotopes or radioactive congeners suitable for nuclear imaging.
photon emission computed tomography (SPECT) or positron One archetypal example of the latter involves 99mTc, which is
emission tomography (PET) is being used to identify the pres- the most widely used radionuclide in diagnostic medicine,
ence of tumors by targeting specific biomarkers, while intrao- where select complexes of technetium’s congener rhenium are
perative fluorescence imaging with the optical properties of luminescent, providing a means of preparing isostructural
multifunctional optical-nuclear probes.28–32
There are a significant number of Re(I) complexes that have
Department of Chemistry and Chemical Biology, McMaster University, 1280 Main
been used as luminescent probes. In particular, mixed ligand
complexes of the [Re(CO)3]+ core derived from bidentate poly-
Street West, Hamilton, Ontario L8S 4 M1, Canada. E-mail: valliant@mcmaster.ca
†Electronic supplementary information (ESI) available: Data include 1H- and
pyridine ligands and an aromatic monodentate ligand, com-
13C-NMR, HRMS, and HPLC UV- and γ-chromatograms; stability study data;
monly referred to as [2 + 1] complexes, have been particularly
absorption and emission spectra. See DOI: 10.1039/d0dt02829k
effective as probes for imaging subcellular structures. These
‡These authors contributed equally.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
complexes have attractive optical properties including broad also prepared to provide a means of further derivatization
absorbance (extending into the visible spectrum) and emission including the addition of a tetrazine to facilitate bioconjuga-
spectra (extending into the near-infrared spectrum), a large tion (vide infra). Characterization data for 3a–3f can be found
Stokes shift, and long fluorescent lifetimes.9,11,33 For many in ESI (Fig. S1–S24†).
luminescent [2 + 1] complexes of this type, the Re chemistry is
well developed, whereas methods for preparing the corres-
ponding 99mTc(I) complexes and studying the chemical and
Preparation and characterization of Re complexes
Prior to radiolabeling studies using 99mTc, the Re(I) complexes
biological properties of the resulting products are notably
lacking.
of the PIP ligands were prepared as reference standards. This
is a necessary step because 99mTc labelling reactions are typi-
cally performed using picomoles of the metal; amounts that
are below the sensitivity of standard characterization methods
(NMR, MS etc.). To address this, the Re complexes are prepared
and characterized and used as HPLC reference standards
where the 99mTc and Re complexes are co-injected and their
retention times (detected by gamma and UV detectors respect-
ively) compared.
Re complexes were synthesized following a literature pro-
cedure.42 PIP ligands (3a–e) were combined with pentacarbo-
nylchlororhenium(I) in toluene and heated the mixture to
reflux for 2 h (Scheme 2). The products (4a–e), were isolated by
HPLC in good yield (average = 78%) where characterization
data (see ESI, Fig. S25–S39†) were consistent with previously
reported values for comparable Re(I) complexes.42
Phenyl imidazole-fused phenanthroline ligands (PIP) have
been widely used to create a range of different luminescent
probes and sensors. These include metal,34–37 ion,38,39 and
singlet oxygen (1O2) sensing constructs.40,41 Yi et al. reported
Re tricarbonyl polypyridine complexes, using imidazole-fused
phenanthroline ligands, which were shown to have strong
absorption bands in the visible region as well as a long-lived
triplet excited state.42 This showed the first use for Re(I) com-
plexes as triplet photosensitizers for triplet–triplet annihil-
ation. To assess the utility of PIP ligands as the basis for creat-
ing isostructural optical-nuclear probes, a method to prepare
99mTc complexes of PIP ligands was developed and the pro-
perties of the radiometal complexes studied. This included
creating and evaluating bifunctional derivatives that can be
linked to targeting molecules using bioorthogonal chemistry.
Compounds 4a–f are unsuitable as molecular imaging
probes because the chloride ligand can be easily substituted
by proteins in the blood such as albumin. For [2 + 1] com-
plexes this has been shown to result in unwanted delivery of
the radiometal complex to the liver, hindering target uptake.11
We previously demonstrated that the extent of non-specific
binding with analogous bipyridine complexes in vivo can be
reduced by replacing the chloride with an N-substituted imid-
azole.11 With this in mind, compounds 4a–e were treated with
N-methylimidazole (NMI) with stirring at room temperature
overnight to form the hitherto unknown PIP complexes 5a–c,e
(Scheme 2). The products were isolated by HPLC in 51 to 80%
yield and characterized by NMR and HRMS (see ESI, Fig. S40–
S51†). The only exception was compound 5d, which did not
form, likely due to the strong electron withdrawing effect of
the nitro group. Similarly, only small amounts of compound
Results and discussion
Ligand synthesis
To assess the impact of substituents on radiochemical yield
and stability of the target 99mTc complexes, a small library of
PIP ligands containing a series of electron donating and with-
drawing groups were prepared. The approach used to syn-
thesize the ligands 3a–f (Scheme 1) was adapted from a litera-
ture procedure42 where 1,10-phenanthroline-5,6-dione, benzal-
dehydes 2a–f and ammonium acetate were added to degassed
glacial acetic acid. After purging with argon, the mixture was
heated to reflux for 3 hours and the desired products collected
in 72 to 93% yield by filtration following the addition of water.
In addition to known PIP derivatives (3a, c–e),43 new ligands
containing a carboxylic acid (3b) and boronic acid (3f) were
Scheme 1 Synthesis of imidazo[4,5-f]-1,10-phenanthroline ligands
3a–f.
Scheme 2 Synthesis of Re-PIP complexes 4a–e and 5a–e.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
Table 1 Absorbance and emission of compounds 4a–e and 5a–f
5e could be isolated, which is also likely due to the electron
withdrawing effect of the cyano group.
4a–f
5a–f
Unfortunately, derivative 5f could not be prepared using
conventional heating due to the poor solubility of the starting
material (3f) in organic solvents. Nevertheless, 5f was desirable
because the boronic acid group provides a convenient site to
use Suzuki–Miyaura coupling chemistry for further ligand
modification or to promote targeting through conjugation to
carbohydrates.44,45 An alternative method using microwave
heating was employed to prepare 5f. In place of [Re(CO)5]Cl,
[Re(CO)3(OH2)3]Br was prepared following the method of
Lazarova et al.44 and added to the PIP ligand and the solution
Absorbancea
(nm)
Emission
(nm)
Absorbance
(nm)
Emission
(nm)
Compound
a: R = H
406
404
412
351
399
—
612
596
616
649
611
—
400
407
414
594
595
574
b: R = COOH
c: R = OMe
d: R = NO2
e: R = CN
b
b
—
—
399
406
611
586
f: R = B(OH)2
a All absorbance and emission data were collected at 50 µM in metha-
nol. Spectra for all compounds are reported in ESI.† b The Re complex
heated in a microwave at 110 °C for 5 min. The NMI ligand 5d was not sufficiently stable to allow for measurement.
was then added to the mixture and heated again at 110 °C for
5 min (Scheme 3). The product was then isolated by HPLC to
afford 5f in 19% yield (see characterization data, Fig. S52–S55
in ESI†). In an attempt to improve the yield, the order of
addition was changed where NMI was combined with [Re
(CO)3(OH2)3]Br and the mixture heated followed by the
addition of PIP ligand. However, there was no further increase
in the yield of 5f. Nonetheless, the amount of product
obtained with the former procedure was sufficient for use as
reference standard for the corresponding 99mTc complex.
Radiochemistry
The 99mTc complexes were prepared using
[
99mTc][Tc
(CO)3(H2O)3]+ as the starting material, which was generated
from [99mTcO4]− following literature methods.46–48 The ligands
(3a–e) (1 mg) were added to a vial containing [99mTc][Tc
(CO)3(H2O)3]+ under argon, and the mixture heated at either 40
or 65 °C for 1 hour (Scheme 4). Radio-HPLC of the reaction
mixtures showed a single peak and no evidence of residual
technetium starting material. Next, the NMI complexes were
prepared by adding the monodentate ligand to compounds
6a–d and heating the mixture for 1 hour at 40 °C. Radio-HPLC
again showed the formation of a single peak at a retention
time that matched that for the corresponding Re analogues.
Compounds 7a–d were isolated free from excess NMI using
HPLC in 57–82% radiochemical yield (see ESI, Fig. S86–S98†).
Compounds containing ligands with electron withdrawing
groups (3d and 3e) showed poor labeling yields comparable to
other derivatives.
Optical properties
The optical properties of the Re complexes were evaluated to
determine the effect that substituents on the PIP ligands have
on the absorption and emission spectra.45 The absorption
spectra for compounds 4a–e showed a broad peak between
351–412 nm, while for 5a–c,e,f the comparable absorption
band appeared between 399–414 nm (Table 1). The corres-
ponding emission spectra appeared between 596–649 nm for
4a–e and 574–632 nm for 5a–c,e,f respectively (full spectra are
provided in ESI, Fig. S63–S82†). The addition of the NMI group
generally caused a blue shift in the absorbance/emission
spectra compared to the precursor compounds containing a
chloride ligand in place of the heterocycle. Electron withdraw-
ing substituents (4b, 4d, 4e) generally were blue shifted relative
to those with electron donating groups on the PIP ligand with
the unsubstituted ring (4a) falling between the two groups as
expected. In general, the values are comparable to literature
data on PIP complexes of Re(I).42
In order to improve the overall efficiency of the synthesis,
attempts were made to develop a one-pot labeling method fol-
lowing an approach reported previously for bipyridine type
ligands.9,11 As a model ligand, 3e was added to a microwave
vial along with [99mTc][Tc(CO)3(H2O)3]+, and an excess of NMI.
The vial was sealed under argon and heated at 40 °C for
Scheme 3 Microwave synthesis of Re-PIP complex 5f.
Scheme 4 Synthesis of 99mTc-labelled PIP complexes 6a–e and 7a–e.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
Tetrazine derivatives
The [2 + 1] PIP complexes can be used to create targeted MI
probes through a number of strategies. This includes the
addition of targeting vectors through the monodentate imid-
azole ligand, which we have described previously for bipyri-
dine complexes,11,27 or by coupling to a functional group on
the bidentate ligand. In the case of the PIP ligands reported
here, the carboxylic acids in compounds 3b can be readily con-
jugated to amino groups using simple active ester chemistry.
This approach can be used to create “actively” targeted MI
probes (i.e. bioconjugation prior to administration), or it can
be employed with pretargeting strategies where coupling
between the tetrazine and strained alkenes can take place
in vivo.49 The use of tetrazines has the added benefit that the
heterocycle can quench the luminescence of the Re complex
creating turn-on type probes.50–52
To this end the PIP ligand 3b was derivatized with the com-
mercially available tetrazine (4-(1,2,4,5-tetrazin-3-yl)phenyl)
methanamine hydrochloride (8), which has been used success-
fully to create radiolabeled tetrazines.27,51,53–56 Coupling was
achieved (Scheme 6) using benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate (PyBOP), where the
desired product (9) was isolated in 68% yield. The thermal sen-
sitivity of tetrazines precluded preparation of the Re reference
standard from 9. As a result, 4b (Re complex of 3b) was
coupled to 8 and the product 10 obtained in 37% yield. The
ligand exchange of the water ligand with NMI was unsuccess-
ful with the Re complex possibly due to the limited tempera-
ture and pH options available that can be used with the highly
reactive tetrazine. The fluorescence spectra of 10 was however
collected and showed an absorbance peak of 425 nm and an
emission peak of 593 nm, where there was a 70-fold increase
in emission intensity (λex = 395 nm) upon the addition of an
excess of (E)-cyclooct-4-enol (TCO-OH) (Fig. 1). The PIP-tetra-
zine ligand was subsequently labeled with 99mTc in a two-step
reaction (Scheme 7). The reaction showed the formation of a
new peak at 12.5 min where the product was isolated in 40%
yield by HPLC which was verified by coinjection with the Re
congener. Addition of NMI (at 40 °C for 1 hour) resulted in a
Scheme 5 One-pot synthesis of 99mTc-labelled 7f.
1 hour. Radio-HPLC confirmed that a single product formed
that had the same HPLC retention time as the product pro-
duced in the two-step labeling, where 7e was obtained in a
55% radiochemical yield (Scheme 5). The same one-pot
method was used to generate 7f. Interestingly, when one-pot
reactions were attempted with rhenium using [Re(CO)3(H2O)3]+
yields were low (<20%) and required higher temperatures
(110 °C) (Scheme 3). Nevertheless, the ability to produce the [2
+ 1] 99mTc complex using a one-pot labelling method at
modest temperatures and times creates the opportunity to
develop instant kits which greatly simplifies getting approval
to use these types of 99mTc complexes in clinical studies.
Stability studies
The stability of the 99mTc complexes (7a–f, and 12) in saline
were monitored by HPLC for 6 to 24 hours. After no evidence
of degradation was noted, the metal complexes were treated
with an excess (2 mM) of cysteine or histidine at 37 °C.
RadioHPLC of the 99mTc complexes again showed no signs of
degradation by 6 hours, which is equivalent to one half-life of
99mTc (see ESI Fig. S85–S101†). These results are consistent
with other [2 + 1] 99mTc(I) complexes containing NMI as the
monodentate ligand.11,27
Scheme 6 Synthesis of tetrazine functionalized PIP ligand 9 and its Re complex 10.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
The TCO-albumin derivative is a convenient tool to test the
reactivity of tetrazines. It may also offer a way to use the
protein to improve solubility and pharmacokinetics of tetra-
zine derived metal complexes. In addition, there is growing
interest in radiolabelled albumins for imaging tumours,
lymph nodes, and infection and as a tool for blood volume
estimation.58,59
Conclusions
A series of phenyl imidazole-fused phenanthroline (PIP)
ligands were synthesized and the corresponding [2 + 1] 99mTc
(I) and Re(I) complexes generated. The Re(I) complexes exhibit
optical properties that are comparable to other Re(I) com-
pounds that have been used as cellular dyes. The 99mTc(I) ana-
logues can be prepared in good yield and under mild con-
ditions, where products were sufficiently stable for use in vivo.
Boronic acid and tetrazine derivatives were prepared allowing
for further functionalization and bioconjugation. In the case
of the tetrazine-PIP derivatives, a turn-on type Re(I) complex
was developed, where the 99mTc analogue can be readily conju-
gated to TCO-modified biomolecules, including TCO-derived
albumin, offering a convenient route to developing targeted
multi-modal probes.
Fig. 1 Top: A scheme illustrating the reaction of Re-tetrazine 10 with
(E)-cyclooct-4-enol (TCO-OH) to produce 11. Bottom left: The emission
spectra (λex = 395 nm) for 10 before (black line) and after (grey line) the
addition of an excess of TCO-OH (50 μM in PBS). Bottom right:
Photograph of 10 under UV light prior to (a) and after (b) treatment with
TCO-OH (sample dissolved in DMSO and diluted to 50 μM with PBS).
Experimental
Materials, instrumentation and general methods
Scheme 7 Two-step radiolabeling of 9.
All reagents and solvents were ACS grade and were purchase
from commercial suppliers and used without further purifi-
cation. (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP) was purchased from Advanced
shift in retention time consistent with compounds 6a–e to 7a–
e. To verify that the product contained the desired tetrazine,
TCO-OH was added, resulting in a small shift in retention time
and change in the number of peaks that is consistent with the
cycloaddition reaction.57 In addition to coupling to a small
molecule TCO derivative, a TCO-derivative of albumin was pre-
pared as a model protein-based targeting molecule. The TCO-
derivative of albumin was prepared by adding trans-4-cyclooc-
ten-1-yl 2,5-dioxo-1-pyrrolidinyl ester carbonic acid (TCO-NHS)
with bovine serum albumin (BSA) at 4 °C for 12 hours.
Following dialysis, the product 14 contained 2.7 TCO groups
per albumin molecule (Fig. S2.63, in the ESI†). When 14 was
added to 99mTc-labelled tetrazine 13 (Scheme 8), complete and
nearly instantaneous consumption of the labelled tetrazine
was evident by radio-HPLC.
ChemTech.
(4-(1,2,4,5-Tetrazin-3-yl)phenyl)methanamine
hydrochloride, and (E)-4-cycloocten-1-yl 2,5-dioxo-1-pyrrolidi-
nyl ester carbonic acid were purchased from Conju-probe.
Toluene, acetone, N,N-dimethylformamide, dimethyl sulfoxide
and methanol were purchased from Caledon. Distilled water
was used for all experiments. Deuterated solvents for NMR
samples were purchased from Cambridge Isotope
Laboratories. Sodium borate and potassium sodium tartrate
were purchased from Anachemia Canada Inc. Sodium carbon-
ate was purchased from EM science. Bovine serum albumin
was purchased from Sigma. Purification performed by column
was done using silica gel 60 (particle size 0.04–0.063 mm) pur-
chased from EMD chemicals. 99mTc was obtained as [99mTc]
−
TcO4 from
a
99Mo/99mTc generator (Lantheus Medical
Imaging) in saline (0.9% NaCl). Caution: 99mTc is a γ-emitter
(Eγ = 140 keV, t1/2 = 6 h) and should only be used in a licensed
and appropriately shielded facility. All other reagents were pur-
chased from Sigma Aldrich.
1H and 13C NMR spectra were recorded on a Bruker Avance
AV-600 instrument at 300 K. High Resolution Mass Spectra
(HRMS) were collected on a Waters/Micromass Q-Tof Global
Ultima spectrometer. BSA samples were analyzed by mass
Scheme 8 Reaction of [99mTc]Tc-tetrazine 13 with TCO-BSA 14.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
spectrometry MALDI Bruker Ultraflextreme spectrometer. H), 9.1 (bs, 2H, Ar–H); 9.0 (m, 4H, Ar–H); 8.4 (bs, 2H, Ar–H);
Microwave reactions were performed using a Biotage Initiator 8.2 (bs, 2H, Ar–H); 7.9 (m, 2H, Ar–H). 13C NMR (DMSO-d6,
60 instrument. Solvents were evaporated using either a 150 MHz): δ (ppm) 172.0, 166.9, 149.4, 148.1, 143.7, 136.0,
Bioatage V10 system or under reduced pressure with a rotary 133.7, 131.3, 130.1, 126.2, 123.7, 123.5, 123.3, 119.3. HRMS:
evaporator. Compounds were dried using a VirTis Benchtop m/z calcd for C20H13N4O2: 341.1039, found: 341.1034. HPLC
lyophilizer equipped with an Edwards RV5 pump. Dialysis was (UV 254 nm): Rt = 6.5 min.
performed using dialysis kit (Spectra/Por 1 Dialysis Tubing,
132 645; 6–8 kDa MWCO).
2-(4-Methoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline,
3c. The ligand was synthesized according to the procedure fol-
High Performance Liquid Chromatography (HPLC) was per- lowed for 3a using 4-methoxybenzaldehyde (52 mg, 0.38 mmol)
formed using a Waters XBridge analytical column (5 µm, 4.6 × in place of benzaldehyde to give a pale yellow solid. Yield: 99 mg
1
100 mm) or C18 preparative column (5 µm, 10 × 100 mm) with (80%). H NMR (CD3OD, 600 MHz): δ (ppm) 13.5 (s, 1H, N–H),
a Waters 1525 system connected to a Bioscan γ detector and a 9.0 (d, 4H, Ar–H); 8.3 (m, 2H, Ar–H); 7.8 (m, 2H, Ar–H); 7.2 (q,
2998 photodiode array detector monitoring at 254 nm operated 2H, Ar–H); 3.8 (s, 4H, -OCH3). 13C NMR (CD3OD, 150 MHz): δ
using the Empower software package. Flow rates were 1 mL (ppm) 160.4, 150.7, 147.6, 143.4, 129.5, 127.8, 123.2, 122.6,
min−1 (analytical) or 4 mL min−1 (preparative) using the fol- 114.4, 55.4. HRMS: m/z calcd for C20H15N4O: 327.1233, found:
lowing methods: Method A: Solvent A: H2O + 0.1% TFA; 327.1233. HPLC (UV 254 nm): Rt = 7.3 min.
Solvent B: Acetonitrile + 0.1% TFA. Gradient: 10% B (0–2 min),
2-(4-Nitrophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, 3d.
100% B (2–12 min), 100% B (12–14 min), 10% B (14–15 min). The ligand was synthesized according to the procedure fol-
Method B: Solvent A: 0.4% ammonium formate (w/v) in H2O; lowed for 3a using 4-nitrobenzaldehyde (57 mg, 0.38 mmol) in
Solvent B: Acetonitrile. Gradient: 10% B (0–2 min), 100% B place of benzaldehyde to give a bright yellow solid. Yield:
(2–12 min), 100% B (12–14 min), 10% B (14–15 min). Method 110 mg (85%). 1H NMR (DMSO-d6, 600 MHz): δ (ppm) 8.99
C: Solvent A: H2O + 0.1% TFA, Solvent B: Acetonitrile + 0.1% (dd, 2H, Ar–H); 8.87 (dd, 2H, Ar–H); 8.47 (dd, 4H, Ar–H); 7.79
TFA. Gradient: 10–70% B (0–14 min), 70% B (14–15 min), (m, 2H, Ar–H). 13C NMR (DMSO-d6, 150 MHz): δ (ppm) 148.3,
70–100% B (15–17 min), 100–10% B (17–18 min). Method D: 147.5, 144.1, 136.2, 129.8, 127.0, 124.5, 123.5. HRMS: m/z calcd
Solvent A: H2O, Solvent B: Acetonitrile. Gradient 10–70% B for C19H11N5O2: 342.0984, found: 342.0984. HPLC (UV
(0–14 min), 70% B (14–15 min), 70–100% B (15–17 min), 254 nm): Rt = 7.7 min.
100–10% B (17–18 min). Method E: Solvent A: H2O, Solvent B:
Acetonitrile. Gradient 10–70% (0–14 min), 70%
2-(4-Cyanophenyl-1H-imidazo[4,5-f][1,10]phenanthroline, 3e.
The ligand was synthesized according to the procedure fol-
B
B
(14–15 min), 70–100% B (15–17 min), 100–10% B (17–18 min), lowed for 3a using 4-formylbenzonitrile (49 mg, 0.38 mmol) in
10% B (18–23 min). Phenomenex PolySep-GFC-P Linear LC place of benzaldehyde to give an orange precipitate. Yield:
Column (300 × 7.8 mm) was also used with the following (76 mg, 93%). TLC Rf = 0.46 (70% DCM/MeOH + 0.01% TFA).
method: Method F: Solvent: H2O. Isocratic, 20 min.
1H NMR (DMSO-d6, 600 MHz): δ (ppm) 14.1 (bs, 1H, Ar–NH);
9.1 (dd, 2H, Ar–H), 9.0 (d, 2H, Ar–H), 8.5 (d, 2H, Ar–H), 8.1 (d,
2H, Ar–H), 7.9 (bs, 2H, Ar–H). 13C NMR (DMSO-d6, 150 MHz):
δ (ppm) 148.2, 143.9, 133.1, 129.7, 126.6, 123.4. HRMS: m/z
Synthetic procedures
2-Phenyl-1H-imidazo[4,5-f][1,10]phenanthroline, 3a. In
a
round bottomed flask, 1,10-phenanthroline-5,6-dione (52 mg, calcd for C20H11N5: 322.3039, found: 322.1084. HPLC (UV
0.25 mmol), benzaldehyde (40 mg, 0.38 mmol) and 254 nm): Rt = 4.4 min.
ammonium acetate (423 mg, 5.50 mmol) were added and the
2-(-Phenyl boronic acid-1H-imidazo[4,5-f][1,10]phenanthro-
flask flushed with Ar for 10 min. To this mixture, degassed line, 3f. In a round bottom flask, 1,10-phelanthroline-5,6-
glacial acetic acid (10 mL) was added and the mixture heated dione (50 mg, 0.24 mmol), 4-formylphenyl boronic acid
at 125 °C for 3 h under Ar. The resulting solution was then (54 mg, 0.36 mmol), and ammonium acetate (403 mg,
cooled, diluted with water (20 mL) and neutralized with con- 5.24 mmol) were combined and the flask flushed with Ar for
centrated NH4OH whereupon a pale peach coloured precipitate 10 min. Degassed glacial acetic acid (10 mL) was added and
formed. The precipitate was collected by filtration, washed the solution heated to reflux (125 °C) under Ar for 3 h. After
with water and dried under vacuum. Yield: 67 mg (89%). 1H cooling to RT, water (20 mL) was added and the resulting pre-
NMR (DMSO-d6, 600 MHz): δ (ppm) 13.7 (s, 1H, Ar–NH); 9.0 cipitate collected by filtration and washed with DI water. The
(dd, 2H, Ar–H); 8.9 (m, 2H, Ar–H); 8.2 (dd, 2H, Ar–H); 7.8 (s, solid was further dried under vacuum yielding a brownish-
2H, Ar–H); 7.6 (m, 2H, Ar–H); 7.5 (m, 1H, Ar–H). 13C NMR yellow solid. Yield: (72 mg, 88%) 1H NMR (DMSO-d6, 600 MHz:
(DMSO-d6, 150 MHz): δ (ppm) 150.6, 147.8, 143.6, 130.2, 129.6, δ (ppm) 13.8 (bs, 1H, Ar–NH), 9.1 (d, 2H, Ar–H), 9.0 (d, 2H, Ar–
129.0, 126.2, 123.3. HRMS: m/z calcd for C19H13N4: 297.1140, H), 8.3 (d, 4H, Ar–H), 8.0 (s, 2H, OH), 7.9 (d, 2H, Ar–H). 13C
found: 297.1153. HPLC (UV 254 nm): Rt = 7.2 min.
NMR (DMSO-d6, 150 MHz: δ (ppm) 150.6, 147.9, 143.7, 135.8,
2-(4-Carboxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline,
134.7, 131.2, 129.7, 125.1, 123.8, 123.5, 123.2, 119.3. HRMS:
3b. The ligand was synthesized according to the procedure fol- m/z calcd for C19H13BN4O2: 341.1213, found: 341.1205. HPLC
lowed for 3a using 4-formylbenzoic acid (57 mg, 0.38 mmol) in (UV 254 nm): Rt = 5.6 min.
place of benzaldehyde, to give a yellow solid. Yield: 93 mg
General synthesis of [Re(CO)3(L)Cl]. To [Re(CO)5Cl] (36 mg,
(72%). 1H NMR (DMSO-d6, 600 MHz): δ (ppm) 14.0 (bs, 1H, N– 0.1 mmol) in toluene (5 ml), the corresponding bidentate
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
1
ligand L (0.12 mmol) in toluene (2 mL) was added and the
[Re(CO)3(4b)(MeIm)]OTf, 5b. H NMR (CD3OD, 600 MHz): δ
mixture heated to reflux for 2 h. A precipitate formed during (ppm) 9.7 (dd, 2H); 9.4 (d, 2H); 8.5 (d, 2H); 8.3 (m, 4H); 7.7 (s,
the course of the reaction, which was collected by filtration 1H); 7.6 (s, 1H); 6.9 (t, 1H); 6.6 (t, 1H); 3.9 (2H); 3.6 (s, 2H). 13
C
and dried under vacuum. The solid was dissolved in DMSO NMR (CD3OD, 150 MHz): δ (ppm) 168.9, 162.2, 154.3, 153.5,
and the desired product isolated by HPLC (Method A) to give a 146.0, 141.9, 134.9, 133.8, 131.6, 129.8, 128.2, 127.9, 123.9,
yellow solid. Yield: 76–80%.
34.5. HRMS: m/z calcd for C26H18N6O3Re: 693.089, found:
[Re(CO)3(3a)Cl], 4a. H NMR (DMSO-d6, 600 MHz): δ (ppm) 693.0889.
14.3 (s, 1H, Ar–NH); 9.4 (d, 2H, Ar–H); 9.3 (d, 2H, Ar–H); 8.3 (d, [Re(CO)3(4c)(MeIm)]OTf, 5c. H NMR (CD3CN, 600 MHz): δ
1
1
2H, Ar–H); 8.2 (bs, 2H, Ar–H); 7.7 (m, 2H, Ar–H); 7.6 (m, 1H, (ppm) 12.1 (s, 1H, Ar–NH); 9.5 (d, 2H, Ar–H); 8.2 (d, 2H, Ar–H);
Ar–H). 13C NMR (DMSO-d6, 150 MHz): δ (ppm) 198.0, 190.3, 8.1 (dd, 2H, Ar–H); 7.2 (s, 1H, Ar–H); 7.2 (d, 2H, Ar–H); 6.8 (s,
151.6, 133.1, 130.6, 129.4, 126.7. HRMS: m/z calcd for 1H, Ar–H); 6.5 (s, 1H, Ar–H); 3.9 (s, 3H, –CH3); 3.4 (s, 3H,
C22H12ClN4O3Re: 601.0069, found: 601.0073.
CH3). 13C NMR (CD3CN, 150 MHz): δ (ppm) 161.4, 153.3,
1
[Re(CO)3(3b)Cl], 4b. H NMR (DMSO-d6, 600 MHz): δ (ppm) 151.8, 151.5, 140.0, 128.4, 128.0, 126.7, 122.2, 121.4, 114.4,
9.4 (d, 2H, Ar–H); 9.3 (d, 2H, Ar–H); 8.5 (d, 2H, Ar–H); 8.2 (d, 55.0, 33.6. HRMS: (ESI+) calcd for C26H18N6O3Re: 679.1105,
2H, Ar–H); 8.2 (q, 2H, Ar–H). 13C NMR (DMSO-d6, 150 MHz): δ found: 679.1087.
1
(ppm) 197.8, 190.0, 166.8, 155.1, 151.5, 144.0, 132.9, 130.7,
[Re(CO)3(4e)(MeIm)]OTf, 5e. H NMR (CD3OD, 600 MHz): δ
126.7, 126.5. HRMS: m/z calcd for C23H12ClN4O5Re: 644.9967, (ppm) 9.5 (dd, 2H); 9.3 (s, 2H); 8.8 (s, 1H); 8.4 (dd, 2H); 8.1 (q,
found: 644.9971. 2H); 7.9 (dd, 2H); 7.6 (s, 1H); 7.5 (d, 2H); 6.8 (t, 1H); 6.4 (t, 1H);
1
[Re(CO)3(3c)Cl], 4c. H NMR (DMSO-d6, 600 MHz): δ (ppm) 3.9 (s, 3H, CH3). 13C NMR (CD3OD, 150 MHz): δ (ppm) 158.1,
14.15 (s, 1H, Ar–NH); 9.37 (dd, 2H, Ar–H); 9.28 (dd, 2H, Ar–H); 140.7, 133.3, 128.3, 127.2, 117.1, 115.4, 109.5, 48.6, 34.0.
8.28 (d, 2H, Ar–H); 8.17 (bs, 2H, Ar–H); 7.23 (m, 2H, Ar–H), HRMS: m/z calcd for C27H17N7O3Re: 674.0952, found:
3.90 (s, 3H, –CH3). 13C NMR (DMSO-d6, 150 MHz): δ (ppm) 674.0943. HPLC (UV 254 nm): Rt = 12.9 min.
197.8, 190.1, 161.0, 152.9, 151.1, 143.5, 132.7, 128.9, 128.2,
126.4, 125.3, 121.9 114.6, 55.4. HRMS: m/z calcd for dissolved in 1 : 1 v/v methanol–water (2 mL). N-Methylimidazole
C23H14ClN4O5Re: 631.0175, found: 631.0192. was then added (0.03 mL, 0.385 mmol) and the mixture heated
Synthesis of 5f. [Re(CO)3(H2O)Br] (25 mg, 0.077 mmol) was
1
[Re(CO)3(3d)Cl], 4d. H NMR (DMSO-d6, 600 MHz): δ (ppm) in a microwave for 5 min at 110 °C. Compound 3f (31 mg,
14.70 (s, 1H, Ar–NH); 9.42 (d, 2H, Ar–H); 9.31 (m, 2H, Ar–H); 0.092 mmol) was added and the solution heated in the micro-
8.57 (m, 4H, Ar–H); 8.21 (bs, 2H, Ar–H). 13C NMR (DMSO-d6, wave for an additional 5 min at 110 °C. The desired product was
150 MHz): δ (ppm) 197.8, 190.1, 152.7, 151.8, 151.5, 132.9, obtained using semi-prep HPLC (Method D) yielding a yellow
1
130.4, 129.3, 127.5, 126.5, 124.6. HRMS: m/z calcd for precipitate. Yield: (10 mg, 19%). H NMR (CD3OD, 600 MHz): δ
C22H11ClN5O5Re: 645.9919, found: 645.9931.
(ppm) 9.57 (dd, 2H), 9.35 (dd, 2H), 8.26 (bs, 2H), 8.19 (q, 2H),
1
[Re(CO)3(3e)Cl], 4e. H NMR (DMSO-d6 600 MHz): δ (ppm) 7.89 (bs, 2H), 7.66 (s, 1H), 6.89 (t, 1H), 6.56 (t, 1H), 3.50 (s, 3H).
14.6 (s, 1H); 9.4 (d, 2H); 9.3 (d, 2H); 8.5 (d, 2H); 8.2 (m, 4H). 13C NMR (CD3OD, 150 MHz): δ (ppm) 161.7, 161.4, 153.4, 145.4,
13C NMR (DMSO-d6, 150 MHz): δ (ppm). 197.6, 189.9, 151.6, 141.8, 135.5, 134.8, 129.8, 127.0, 125.4, 123.9, 34.5. HRMS: m/z
150.5, 144.0, 141.1, 133.2, 133.1, 132.8, 132.7, 129.0, 128.3, calcd for C26H19BN6O5Re: 693.1073, found: 693.1097. HPLC (UV
126.9, 126.6, 118.4, 112.1. HRMS: m/z calcd for 254 nm): Rt = 11.2 min.
C23H11ClN5O3Re: 626.0021, found: 626.0008.
Synthesis of tetrazine-functionalized PIP ligand, 9. (4-
(1,2,4,5-Tetrazin-3-yl)phenyl)methanamine hydrochloride (42 mg,
0.19 mmol) was dissolved in DMF (2 mL) and DIPEA (48 mg,
0.37 mmol) was added and the resulting solution stirred for
General method for the preparation of [Re(CO)3(L)(MeIm)]OTf
[Re(CO)3(L)Cl] (30 mg, 0.05 mmol) in acetone (5 mL) was 15 min. The solution was then added 3b (38 mg, 0.11 mmol) and
mixed with AgOTf (12.8 mg, 0.05 mmol) in 1 mL acetone, and PyBOP (105 mg, 0.2 mmol) in DMF (2 mL) and the mixture
the mixture heated at 70 °C for 2 h. A precipitate formed stirred for 2 h. Water (20 mL) was added and the resulting pre-
during the course of the reaction, which was collected by fil- cipitate was collected by centrifugation, which was re-dissolved in
tration. To the filtrate, NMI (16 mg, 0.2 mmol) was added at DMSO and the desired product isolated as a pink powder by
room temperature and the mixture stirred overnight. The HPLC (Method C). Yield: 38 mg (68%). 1H NMR (DMSO-d6,
solvent was removed by rotary evaporation and the resulting 700 MHz): δ (ppm) 10.57 (s, 1H); 9.41 (t, 1H); 9.31 (d, 2H); 9.16
solid was dissolved in DMSO and the desired product obtained (dd, 2H); 8.48 (d, 2H); 8.35 (d, 2H); 8.23 (m, 2H); 8.15 (d, 2H);
by HPLC (Method A) as a yellow solid. Yield: 51–80%.
7.65 (d, 2H); 4.67 (d, 2H). 13C NMR (DMSO-d6, 176 MHz): δ (ppm)
1
[Re(CO)3(4a)(Melm)]OTf, 5a. H NMR (CD3OD, 600 MHz): δ 166.01, 159.15, 158.94, 158.73, 158.56, 151.72, 145.78, 145.33,
(ppm) 9.6 (dd, 2H, Ar–H); 9.3 (dd, 2H, Ar–H); 8.2 (d, 2H, Ar– 135.73, 132.05, 130.88, 128.57, 126.74, 126.23, 43.11. HRMS: m/z
H); 8.2 (q, 2H, Ar–H); 7.7 (s, 1H, Ar–H); 7.6 (md, 3H, Ar–H); 6.9 calcd for C29H19N9O: 510.17, found: 510.1785.
(s, 1H, Ar–H); 6.6 (s, 1H, Ar–H); 3.5 (s, 3H, –CH3). 13C NMR
Tetrazine-functionalized PIP Re complex, 10. (4-(1,2,4,5-
(DMSO-d6, 150 MHz): δ (ppm) 150.0, 142.2, 140.5, 133.2, 128.5, Tetrazin-3-yl)phenyl)methanamine hydrochloride (19 mg,
128.0, 126.4, 126.1, 122.9, 33.9. HRMS: m/z calcd for 0.1 mmol) was dissolved in DMF (1 mL) and DIPEA (26 mg,
C26H18N6O3Re: 649.0999, found: 649.0994.
0.2 mmol) was added and the resulting solution stirred for
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
15 min. The solution was added 5b (32 mg, 0.05 mmol) and
One-pot synthesis of 7f. Compound 3f (1 mg), NMI (50 μL)
PyBOP (52 mg, 0.1 mmol) in DMF (1 mL) and the mixture and [99mTc(CO)3(OH2)3]+ (2 mL) were added to a microwave
stirred for 2 h. Water (20 mL) was added and the resulting pre- vial, sealed, and heated to 40 °C under Ar for 1 h. The mixture
cipitate collected by centrifugation, dissolved in DMSO and was analyzed for purity using analytical HPLC to confirm for-
the desired product isolated as an orange powder by HPLC mation of the product. Rt = 11.3 min, RCY = 49% (n = 2).
(Method C). Yield: 30 mg (37%). 1H NMR (DMSO-d6,
Two-step synthesis of 13. Compound 9 (41 mg) and [99mTc
600 MHz): δ (ppm) 10.60 (s, 1H, Ar–H); 9.40 (dd, 3H, Ar–H); (CO)3(OH2)3]+ (2 mL) were added to a microwave vial, sealed,
9.33 (dd, 2H, Ar–H); 8.52 (d, 2H, Ar–H); 8.46 (d, 2H, Ar–H); and heated to 40 °C under Ar for 3 h. The product 12 was puri-
8.23 (d, 3H, Ar–H); 7.68 (d, 2H, Ar–H); 4.70 (s, 2H, –CH2–). 13C fied from the reaction mixture via HPLC (Method E Rt
=
NMR (DMSO-d6, 176 MHz): δ (ppm) 198.31, 190.47, 166.28, 12.5 min, RCY = 35% (n = 2). To the purified final product 13,
165.88, 158.62, 152.22, 151.98, 145.26, 144.47, 135.83, 133.36, NMI (50 μL) was added and heated to 40 °C under Ar for 1 h
132.26, 130.91, 128.73, 128.37, 127.14, 126.87. HRMS: m/z (Method E Rt = 13 min, RCY = 24% (n = 1).
calcd for C32H19ClN9O4Re: 814.0720, found: 814.0737 (with Re
+ Cl isotope pattern).
TCO-functionalized BSA, 14. trans-4-Cycloocten-yl 2,5-dioxo-
1-pyrrolidinyl ester carbonic acid (9.3 mg, 0.061 mmol) was
Conflicts of interest
dissolved in DMSO (0.2 mL), and the solution added to bovine There are no conflicts to declare.
serum albumin (200 mg, 0.003 mmol) in water (2 mL). The
reaction mixture was left to shake at 4 °C for 12 h. The product
was then purified via dialysis against MilliQ water for 12 h.
The product was obtained following lyophilization as a white
Acknowledgements
solid. It was determined by MALDI-TOF MS that there were 2.7 The authors gratefully acknowledge funding support from the
TCO per BSA molecule.
Natural Science and Engineering Research Council (NSERC,
#227514) of Canada, Canadian Cancer Society (CCS, #703857),
the Canadian Foundation of Innovation (CFI, #35354) and the
Radiochemistry
General synthesis of [99mTc(CO)3(L)Cl] and [99mTc(CO)3(L) Ontario Institute for Cancer Research (OICR, #C. SI.015.8). The
NMI]Cl. Sodium boranocarbonate (10.0 mg, 0.07 nmol), authors acknowledge the contributions of Dr Denis Snider,
sodium carbonate (15.0 mg, 14.0 mmol), sodium borate who provided scientific proofreading and editing in the prepa-
(20 mg, 0.05 mmol) and sodium potassium tartrate (22 mg, ration of this manuscript, and Dr Afaf Genady and Dr Jeff Kent
0.08 mmol) were placed in a microwave vial and purged with for their help with synthesis.
argon for 10 min. To this mixture, Na[99mTcO4] (1 mL, 1.85
GBq) was added and the vial was heated in a microwave at
110 °C for 3.5 min to form [99mTc(CO)3(H2O)3]+. After cooling
to room temperature, the pH was adjusted to 2 with 0.1 M HCl
References
(0.2 mL) and the solution added to 3a–d (1 mg, 6 mmol) under
argon. The solution was stirred at 40 or 65 °C for 1 h where the
formation of 6a–d was confirmed by analytical HPLC (Method
B) and through comparison of retention times of the co-
injected Re analogue. 6a: Rt = 10.2 min, RCY = 63% (n = 3); 6b:
Rt = 7.9 min, RCY = 82% (n = 3); 6c: Rt = 10.4 min, RCY = 57%
(n = 2); 6d: Rt = 10.2 min, RCY = 64% (n = 2). The
N-methylimidazole (NMI) derivatives 6a–d were prepared by
adding NMI (50 μL, 0.75 mmol) to the solution containing 6a–
d under argon where the mixture was stirred at 40 °C for 1 h.
The formation of products 7a–d was confirmed was confirmed
by analytical HPLC (Method B) and through comparison of
1 S. M. Ametamey, M. Honer and P. A. Schubiger, Molecular
imaging with PET, Chem. Rev., 2008, 108(5), 1501–1516.
2 H. R. Herschman, Molecular imaging: looking at problems,
seeing solutions, Science, 2003, 302(5645), 605–608.
3 J. Kim, Y. Piao and T. Hyeon, Multifunctional nano-
structured materials for multimodal imaging, and simul-
taneous imaging and therapy, Chem. Soc. Rev., 2009, 38(2),
372–390.
4 J. C. Paeng and D. S. Lee, Multimodal Molecular Imaging
In Vivo, Open Nucl. Med. J., 2010, 2, 145–152.
5 F. L. Thorp-Greenwood and M. P. Coogan, Multimodal
radio- (PET/SPECT) and fluorescence imaging agents based
on metallo-radioisotopes: current applications and pro-
spects for development of new agents, Dalton Trans., 2011,
40(23), 6129–6143.
retention times of the co-injected Re analogue. 7a: Rt
=
10.7 min, RCY = 77% (n = 3); 7b: Rt = 8.3 min, RCY = 89% (n =
3); 7c: Rt = 9.7 min, RCY = 66% (n = 3); 7d: Rt = 10.7 min, RCY
= 54% (n = 3).
One-pot synthesis of 7e. Compound 3e (1 mg), NMI (50 μL)
and [99mTc(CO)3(OH2)3]+ (2 mL) were added to a microwave
vial, sealed, and heated to 40 °C under Ar for 1 h. The mixture
was analyzed for purity using analytical HPLC Method C to
confirm formation of the product. Rt = 13.1 min, RCY = 55%
(n = 2).
6 K. R. Bhushan, P. Misra, F. Liu, S. Mathur, R. E. Lenkinski
and J. V. Frangioni, Detection of breast cancer microcalcifi-
cations using
a dual-modality SPECT/NIR fluorescent
probe, J. Am. Chem. Soc., 2008, 130(52), 17648–17649.
7 L. E. Jennings and N. J. Long, ‘Two is better than one’–
probes for dual-modality molecular imaging, Chem.
Commun., 2009, (24), 3511–3524.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
8 G. R. Morais, A. Paulo and I. Santos, Organometallic 21 S. S. Li, C. Zhang, S. Y. Huang, F. Hu, J. Yin and S. H. Liu,
Complexes for SPECT Imaging and/or Radionuclide
Therapy, Organometallics, 2012, 31(16), 5693–5714.
9 T. S. Pitchumony, L. Banevicius, N. Janzen, J. Zubieta and
J. F. Valliant, Isostructural nuclear and luminescent probes
Highly selective colorimetric and fluorescent sensors for
the fluoride anion based on imidazo[4,5-f]-1,10-phenan-
throline metal-complexes, RSC Adv., 2012, 2(10), 4215–
4219.
derived from stabilized [2 + 1] rhenium(I)/technetium(I) 22 S. Liu, T. P. Lin, D. Li, L. Leamer, H. Shan, Z. Li,
organometallic complexes, Inorg. Chem., 2013, 52(23),
13521–13528.
10 C. Spagnul, R. Alberto, G. Gasser, S. Ferrari, V. Pierroz,
A. Bergamo, T. Gianferrara and E. Alessio, Novel water-
soluble (99 m)Tc(I)/Re(I)-porphyrin conjugates as potential
F. P. Gabbai and P. S. Conti, Lewis acid-assisted isotopic
18F-19F exchange in BODIPY dyes: facile generation of
positron emission tomography/fluorescence dual modality
agents for tumor imaging, Theranostics, 2013, 3(3), 181–
189.
multimodal agents for molecular imaging, J. Inorg. 23 T. Priem, C. Bouteiller, D. Camporese, X. Brune,
Biochem., 2013, 122, 57–65.
J. Hardouin, A. Romieu and P. Y. Renard, A novel sulfo-
nated prosthetic group for [18F]-radiolabelling and impart-
ing water solubility of biomolecules and cyanine fluoro-
phores, Org. Biomol. Chem., 2013, 11(3), 469–479.
11 A. Yazdani, N. Janzen, L. Banevicius, S. Czorny and
J. F. Valliant, Imidazole-based [2 + 1] Re(I)/99mTc(I) com-
plexes as isostructural nuclear and optical probes, Inorg.
Chem., 2015, 54(4), 1728–1736.
12 M. C. Boonstra, J. Prakash, C. J. Van De Velde,
W. E. Mesker, P. J. Kuppen, A. L. Vahrmeijer and C. F. Sier,
Stromal Targets for Fluorescent-Guided Oncologic Surgery,
Front. Oncol., 2015, 5, 254.
24 R. Ting, T. A. Aguilera, J. L. Crisp, D. J. Hall,
W. C. Eckelman, D. R. Vera and R. Y. Tsien, Fast 18F label-
ing of a near-infrared fluorophore enables positron emis-
sion tomography and optical imaging of sentinel lymph
nodes, Bioconjugate Chem., 2010, 21(10), 1811–1819.
13 S. Miwa, Y. Matsumoto, Y. Hiroshima, S. Yano, F. Uehara, 25 R. Yan, E. El-Emir, V. Rajkumar, M. Robson, A. P. Jathoul,
M. Yamamoto, Y. Zhang, H. Kimura, K. Hayashi,
N. Yamamoto, M. Bouvet, N. Sugimoto, H. Tsuchiya and
R. M. Hoffman, Fluorescence-guided surgery of prostate
cancer bone metastasis, J. Surg. Res., 2014, 192(1), 124–133.
R. B. Pedley and E. Arstad, One-Pot Synthesis of an I-125-
Labeled Trifunctional Reagent for Multiscale Imaging with
Optical and Nuclear Techniques, Angew. Chem., Int. Ed.,
2011, 50(30), 6793–6795.
14 S. B. Mondal, S. Gao, N. Zhu, R. Liang, V. Gruev and 26 Y. Zhou, Y. S. Kim, X. Yan, O. Jacobson, X. Chen and S. Liu,
S. Achilefu, Real-Time Fluorescence Image-Guided
Oncologic Surgery, Adv. Cancer Res., 2014, 124, 171–211.
15 Q. T. Nguyen and R. Y. Tsien, Fluorescence-guided surgery
64Cu-labeled lissamine rhodamine B: a promising PET
radiotracer targeting tumor mitochondria, Mol. Pharm.,
2011, 8(4), 1198–1208.
with live molecular navigation–a new cutting edge, Nat. 27 A. Yazdani, N. Janzen, S. Czorny, R. G. Ungard,
Rev. Cancer, 2013, 13(9), 653–662.
T. Miladinovic, G. Singh and J. F. Valliant, Preparation of
tetrazine-containing [2 + 1] complexes of 99mTc and
in vivo targeting using bioorthogonal inverse electron
demand Diels-Alder chemistry, Dalton Trans., 2017, 46,
14691–14699.
16 A. L. Vahrmeijer, M. Hutteman, J. R. van der Vorst,
C. J. van de Velde and J. V. Frangioni, Image-guided cancer
surgery using near-infrared fluorescence, Nat. Rev. Clin.
Oncol., 2013, 10(9), 507–518.
17 V. Duheron, M. Moreau, B. Collin, W. Sali, C. Bernhard, 28 B. B. Azad, C. F. Cho, J. D. Lewis and L. G. Luyt, Synthesis,
C. Goze, T. Gautier, J. P. Pais de Barros, V. Deckert,
F. Brunotte, L. Lagrost and F. Denat, Dual labeling of lipo-
radiometal labeling and in vitro evaluation of a targeted
PPIX derivative, Appl. Radiat. Isot., 2012, 70(3), 505–511.
polysaccharides for SPECT-CT imaging and fluorescence 29 S. Botchway, J. R. Dilworth and M. Salichou, One and two
microscopy, ACS Chem. Biol., 2014, 9(3), 656–662.
photon fluorescent complexes of rhenium and their tech-
18 S. C. Ghosh, P. Ghosh, N. Wilganowski, H. Robinson,
netium analogues, Dalton Trans., 2010, 39(22), 5219–5220.
M. A. Hall, G. Dickinson, K. L. Pinkston, B. R. Harvey, 30 T. U. Connell, D. J. Hayne, U. Ackermann, H. J. Tochon-
E. M. Sevick-Muraca and A. Azhdarinia, Multimodal chela-
tion platform for near-infrared fluorescence/nuclear
imaging, J. Med. Chem., 2013, 56(2), 406–416.
Danguy, J. M. White and P. S. Donnelly, Rhenium and tech-
netium tricarbonyl complexes of 1,4-Substituted pyridyl-
1,2,3-triazole bidentate ‘click’ ligands conjugated to a tar-
geting RGD peptide, J. Labelled Compd. Radiopharm., 2014,
57(4), 262–269.
19 T. K. Heinrich, V. Gottumukkala, E. Snay, P. Dunning,
F. H. Fahey, S. Ted Treves and A. B. Packard, Synthesis of
fluorine-18 labeled rhodamine B: A potential PET myocar- 31 S. Faulkner and N. J. Long, Radiopharmaceuticals for
dial perfusion imaging agent, Appl. Radiat. Isot., 2010,
68(1), 96–100.
imaging and therapy, Dalton Trans., 2011, 40(23), 6067–
6067.
20 J. A. Hendricks, E. J. Keliher, D. Wan, S. A. Hilderbrand, 32 E. Ranyuk, R. Lebel, Y. Berube-Lauziere, K. Klarskov,
R. Weissleder and R. Mazitschek, Synthesis of [18F]
BODIPY: bifunctional reporter for hybrid optical/positron
emission tomography imaging, Angew. Chem., Int. Ed.,
2012, 51(19), 4603–4606.
R. Lecomte, J. E. van Lier and B. Guerin, (68)Ga/DOTA- and
(64)Cu/NOTA-phthalocyanine conjugates as fluorescent/
PET bimodal imaging probes, Bioconjugate Chem., 2013,
24(9), 1624–1633.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
33 K. K. Lo, A. W. Choi and W. H. Law, Applications of lumi- 46 R. Alberto, K. Ortner, N. Wheatley, R. Schibli and
nescent inorganic and organometallic transition metal
complexes as biomolecular and cellular probes, Dalton
Trans., 2012, 41(20), 6021–6047.
A. P. Schubiger, Synthesis and Properties of
Boranocarbonate: A Convenient in Situ CO Source for the
Aqueous Preparation of [99mTc(OH2)3(CO)3]+, J. Am. Chem.
Soc., 2001, 123(13), 3135–3136.
34 M. Alfonso, A. Tarraga and P. Molina, Ferrocene-based het-
eroditopic receptors displaying high selectivity toward lead 47 R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, U. Abram
and mercury metal cations through different channels,
J. Org. Chem., 2011, 76(3), 939–947.
35 N. Kaur and P. Alreja, A novel 1,10-phenanthroline based
chemosensor for differential metal ion sensing and con-
structing molecular logic gates, Tetrahedron Lett., 2015,
56(1), 182–186.
36 M. J. E. Resendiz, J. C. Noveron, H. Disteldorf, S. Fischer
and P. J. Stang, A self-assembled supramolecular optical
sensor for Ni(II), Cd(II), and Cr(III), Org. Lett., 2004, 6(5),
651–653.
and T. A. Kaden, A Novel Organometallic Aqua Complex of
Technetium for the Labeling of Biomolecules: Synthesis of
[
99mTc(OH2)3(CO)3]+ from [99mTcO4]− in Aqueous Solution
and Its Reaction with a Bifunctional Ligand, J. Am. Chem.
Soc., 1998, 120(31), 7987–7988.
48 R. W. Simms, P. W. Causey, D. M. Weaver, C. Sundararajan,
K. A. Stephenson and J. F. Valliant, Preparation of techne-
tium-99 m bifunctional chelate complexes using a micro-
fluidic reactor: a comparative study with conventional and
microwave labeling methods, J. Labelled Compd.
Radiopharm., 2012, 55(1), 18–22.
37 D. Sun, Y. Liu, Q. Yu, Y. Zhou, R. Zhang, X. Chen, A. Hong
and J. Liu, The effects of luminescent ruthenium(II) poly- 49 R. Rossin, S. M. van den Bosch, W. Ten Hoeve, M. Carvelli,
pyridyl functionalized selenium nanoparticles on bFGF-
induced angiogenesis and AKT/ERK signaling,
Biomaterials, 2013, 34(1), 171–180.
38 M. Hardouin-Lerouge, B. Chesneau, M. Allain and
R. M. Versteegen, J. Lub and M. S. Robillard, Highly reac-
tive trans-cyclooctene tags with improved stability for Diels-
Alder chemistry in living systems, Bioconjugate Chem.,
2013, 24(7), 1210–1217.
P. Hudhomme, Dual signaling system with an extended-tet- 50 A. W. Choi, K. K. Tso, V. M. Yim, H. W. Liu and K. K. Lo,
rathiafulvalene-phenanthroline dyad acting as an electroop-
tical cation chemosensor, J. Org. Chem., 2012, 77(5), 2441–
2445.
Modification of 1,2,4,5-tetrazine with cationic rhenium(I)
polypyridine units to afford phosphorogenic bioorthogonal
probes with enhanced reaction kinetics, Chem. Commun.,
2015, 51(16), 3442–3445.
39 C. G. Wu, H. C. Lu, L. N. Chen and Y. C. Lin, Synthesis and
ion-sensing phenomena of two new helical conjugated oli- 51 K. K. Lo, K. H. Tsang, W. K. Hui and N. Zhu, Luminescent
gomers containing 1,10-phenanthroline and oligo-
alkylthiophene, J. Polym. Sci., Part A: Polym. Chem., 2008,
46(5), 1586–1597.
rhenium(I) diimine indole conjugates–photophysical,
electrochemical and protein-binding properties, Chem.
Commun., 2003, (21), 2704–2705.
40 L. F. Shi and B. Li, A Series of Cu-I Complexes Containing 52 A. W.-T. Choi, K. K.-S. Tso, V. M.-W. Yim,
1,10-Phenanthroline
Characterization, Photophysical, and Oxygen-Sensing
Properties, Eur. J. Inorg. Chem., 2009, (15), 2294–2302.
Derivative
Ligands:
Synthesis,
H.-W. Liu and K. K.-W. Lo, Modification of 1,2,4,5-
Tetrazine with Cationic Rhenium(I) Polypyridine Units to
Afford Phosphorogenic Bioorthogonal Probes with
Enhanced Reaction Kinetics, Chem. Commun., 2015, 51(16),
3442–3445.
41 H. J. Yin, Y. J. Liu, J. Gao and K. Z. Wang, A highly sensitive
and selective visible-light excitable luminescent probe for
singlet oxygen based on a dinuclear ruthenium complex, 53 H. A. Bilton, Z. Ahmad, N. Janzen, S. Czorny and
Dalton Trans., 2017, 46(10), 3325–3331.
42 X. Yi, J. Zhao, W. Wu, D. Huang, S. Ji and J. Sun, Rhenium
(I) tricarbonyl polypyridine complexes showing strong
J. F. Valliant, Preparation and Evaluation of 99mTc-Labeled
Tridentate Chelates for Pre-Targeting Using Bioorthogonal
Chemistry, J. Vis. Exp., 2017, 120, e55188.
absorption of visible light and long-lived triplet excited 54 N. K. Devaraj and R. Weissleder, Biomedical Applications
states as a triplet photosensitizer for triplet-triplet annihil-
ation upconversion, Dalton Trans., 2012, 41(29), 8931–8940.
Fo Tetrazine Cycloadditions, Acc. Chem. Res., 2011, 44(9),
816–827.
43 S. S. Qasim, S. S. Ali and S. K. Ahmed, SnCl2.2H2O cata- 55 B. Zeglis, K. K. Sevak, T. Reiner, P. Mohindra,
lyzed one-pot synthesis of 2-phenylimidazo [4, 5-f] [1, 10]
phenanthroline, Res. J. Pharm., Biol. Chem. Sci., 2011, 2(2),
423–428.
S. D. Carlin, P. Zanzonico, R. Weissleder and J. S. Lewis, A
Pretargeted PET Imaging Strategy Based on Bioorthogonal
Diels-Alder Click Chemistry, J. Nucl. Med., 2013, 54(8),
1389–1396.
44 N. Lazarova, S. James, J. Babich and J. Zubieta, A con-
venient synthesis, chemical characterization and reactivity 56 J. P. Meyer, P. Kozlowski, J. Jackson, K. M. Cunanan,
of [Re(CO)3(H2O)3]Br: the crystal and molecular structure
of [Re(CO)3(CH3CN)2Br], Inorg. Chem. Commun., 2004,
7(9), 1023–1026.
P. Adumeau, T. R. Dilling, B. M. Zeglis and J. S. Lewis,
Exploring Structural Parameters for Pretargeting
Radioligand Optimization, J. Med. Chem., 2017, 60(19),
8201–8217.
45 S. Abou-Hatab, V. A. Spata and S. Matsika, Substituent
Effects on the Absorption and Fluorescence Properties of 57 M. L. Blackman, M. Royzen and J. M. Fox, The Tetrazine
Anthracene, J. Phys. Chem. A, 2017, 121(6), 1213–1222.
Ligation: Fast Bioconjugation Based on Inverse-Electron-
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
Demand Diels-Alder Reactivity NIH Public Access, J. Am.
Chem. Soc., 2008, 130(41), 13518–13519.
Radiolabeled Monoclonal Antibody Technique, Infect.
Immun., 2001, 69(5), 3460–3465.
58 H. C. Van Der Heyde, P. Bauer, G. Sun, W. L. Chang, L. Yin, 59 L. Yao, X. Xue, P. Yu, Y. Ni and F. Chen, Evans Blue Dye: A
J. Fuseler and D. N. Granger, Assessing Vascular
Permeability during Experimental Cerebral Malaria by a
Revisit of Its Applications in Biomedicine, Contrast Media
Mol. Imaging, 2018, 2018, 18–24.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.