Detecting hypoxia: In vitro using 18F-pretargeted IEDDA click chemistry in live cells

Article abstract of DOI:10.1039/d1ra02482e

We have exemplified a pretargeted approach to interrogate hypoxia in live cells using radioactive bioorthogonal inverse electron demand Diels-Alder (IEDDA) click chemistry. Our novel 18F-tetrazine probe ([18F]FB-Tz) and 2-nitroimidazole-based TCO targeting molecule (8) showed statistically significant (P < 0.0001) uptake in hypoxic cells (ca. 90 %ID per mg) vs. normoxic cells (<10 %ID per mg) in a 60 min incubation of [18F]FB-Tz. This is the first time that an intracellularly targeted small-molecule for IEDDA click has been used in conjunction with a radioactive reporter molecule in live cells and may be a useful tool with far-reaching applicability for a variety of applications. This journal is

Full text of DOI:10.1039/d1ra02482e

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PAPER
1
8
Detecting hypoxia in vitro using F-pretargeted
IEDDA “click” chemistry in live cells†
Cite this: RSC Adv., 2021, 11, 20335
ab
a
a
a
a
Louis Allott,
Cen Chen, Marta Braga, Sau Fung Jacob Leung, Ning Wang,
a
a
ac
a
Chris Barnes, Diana Brickute, Laurence Carroll
and Eric O. Aboagye
*
We have exemplified a pretargeted approach to interrogate hypoxia in live cells using radioactive
18
bioorthogonal inverse electron demand Diels–Alder (IEDDA) “click” chemistry. Our novel F-tetrazine
18
probe ([ F]FB-Tz) and 2-nitroimidazole-based TCO targeting molecule (8) showed statistically significant
Received 29th March 2021
Accepted 1st June 2021
(
P < 0.0001) uptake in hypoxic cells (ca. 90 %ID per mg) vs. normoxic cells (<10 %ID per mg) in a 60 min
18
incubation of [ F]FB-Tz. This is the first time that an intracellularly targeted small-molecule for IEDDA
click” has been used in conjunction with a radioactive reporter molecule in live cells and may be
a useful tool with far-reaching applicability for a variety of applications.
DOI: 10.1039/d1ra02482e
“
rsc.li/rsc-advances
reporter molecules may overcome the challenges associated
with quantifying a uorescence signal by microscopy and
Introduction
An ever-growing toolbox of “click” chemistries offers rapid, negate the physical limitations of some uorophores, such as
1
14
chemoselective ligation in aqueous reaction milieu. The photobleaching.
inverse electron demand Diels–Alder (IEDDA) “click” reaction
We report a pretargeted IEDDA “click” method for detecting
between tetrazine (Tz) and trans-cyclooctene (TCO) under hypoxia in cancer cells with a small molecule 2-nitroimidazole
4
5
ꢀ1 ꢀ1
18
physiological conditions (10 –10
M
s
) has been heavily targeting vector and a cell-permeable F-radiolabelled tetra-
investigated by the research community, particularly for per- zine. The 2-nitroimidazole pharmacophore accumulates in
1
–5
forming bioorthogonal reactions. The commercial availability hypoxic cells via a well-known redox mechanism (Fig. 1); rstly,
of Tz and TCO intermediates has facilitated the rapid and the compound diffuses passively across the cell membrane and
convenient assembly of complex conjugates and has conve- the nitro-group is enzymatically reduced to a reactive species
niently placed biorthogonal chemistry in the hands of scien- inside the cell. Under normoxic conditions, the nitro species is
tists. This technology has been used to visualise and quantify re-oxidized and can diffuse out of the cell. Under hypoxic
intracellular and extracellular targets by live cell uorescence conditions, the nitro species is further reduced to a nitroso
imaging and the medical imaging community has developed moiety and ultimately to a primary amine; the reactive inter-
a new eld of pretargeted positron emission tomography (PET) mediates bind covalently to intracellular macromolecules and
15
imaging for the in vivo detection of TCO-modied monoclonal are thereby trapped. Fluorine-18 PET radiotracers based
6
–11
antibodies (mAb). The study of in vitro intracellular targets in around the 2-nitroimidazole pharmacophore have been devel-
1
8
18
live cells has predominantly focused on uorescence imaging, oped (i.e. [ F]uoromisonidazole, [ F]FMISO, Fig. 1C) but
where a functionalised biomolecule bearing a tetrazine-reactive their cellular uptake is low, owed to the difference between the
handle (e.g. TCO) accumulates within a cell which bio- physical half-life of the isotope and the time required for
12,13
orthogonally reacts with a uorescent-Tz reporter molecule;
surprisingly, the use of radioactive reporter molecules in live (ca. 3–4 h).
sufficient retention by the aforementioned redox mechanism
16–20
By using a pretargeted approach, we hypoth-
cells has not been reported. Detecting radioactive species is esised that a 2-nitroimidazole pharmacophore bearing a TCO
a highly sensitive and quantitative technique, and radiolabelled group, when allowed to accumulate inside the cell over a longer
1
8
period of time (12 h) with follow-up incubation of an F-
tetrazine (60 min incubation), may give better sensitivity of
detection and higher in vitro accumulation than is possible with
conventional radiotracers (Fig. 1).
a
Comprehensive Cancer Imaging Centre, Faculty of Medicine, Department of Surgery
and Cancer, Imperial College London, Hammersmith Hospital, Du Cane Road,
London, W12 0NN, UK. E-mail: eric.aboagye@imperial.ac.uk
b
Positron Emission Tomography Research Centre, Faculty of Health Sciences,
University of Hull, Cottingham Road, Kingston upon Hull, HU6 7RX, UK
c
Results & discussion
Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins
Medical Institutions, Baltimore, Maryland, USA
A simple TCO-modied 2-nitroimidazole was required for the in
vitro pretargeted detection of hypoxia. (E)-Cyclooct-3-en-1-yl(2-
†
Electronic supplementary information (ESI) available: Compound
characterisation data. See DOI: 10.1039/d1ra02482e
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bioorthogonal IEDDA “click’ chemistry (Scheme 1B). Firstly, 2-
nitroimidazole (5) was reacted with 2-(boc-amino)ethyl bromide
to install the short alkyl chain linker (6). The boc-protecting
group was hydrolysed under acidic conditions, producing
a primary alkylammonium salt (7) which was quenched under
basic conditions followed by the addition of TCO-NHS ester to
produce compound 8 in a 53% yield.
1
8
18
A cell-permeable F-tetrazine ([ F]FB-Tz) was synthesised
which was required to passively diffuse across the cell
membrane of all cells without discrimination, with selective
1
8
retention of [ F]FB-Tz in cells containing the retained TCO
moiety (Scheme 1); this predicates a lipophilic tetrazine
(
log D7.5 > 1). Using simple reductive amination radiochemistry
1
8
18
and the 4-[ F]uorobenzaldehyde ([ F]FBA) prosthetic group
with a previously reported automated radiolabelling method-
ology developed in our group on the GE FASTLab™ platform
1
8
(Fig. 2A), we synthesised [ F]FB-Tz from precursor 3 (Scheme
1
8
1
C). [ F]FB-Tz was produced in a 8.7 ꢁ 1.1 non-decay corrected
radiochemical yield (RCY n.d.c), a molar activity of 5 GBq
mmol and >96% radiochemical purity in 80 min (Fig. 2C and
D). [ F]FB-Tz was lipophilic with an experimental log D7.5 of
ꢀ
1
21
18
1
.14 ꢁ 0.03.
With both the targeting molecule (8) and the radioactive
1
8
reporter molecule ([ F]FB-Tz) in hand, the approach was eval-
uated biologically in hypoxic and normoxic cancer cell lines.
Fig. 1 (A) Mechanism of 2-nitroimidazole retention in hypoxic cells
exemplified by a TCO-containing derivative for intracellular pretar-
geted detection; (B) detecting the accumulation and retention of the
TCO-bearing targeting molecule in hypoxic cells using a cell perme-
able radiolabelled tetrazine. (C) Structure of a fluorine-18 radiolabelled
Biological evaluation in live cells
18
hypoxia radiotracer ([ F]FMISO).
1
8
Compound [ F]FB-Tz and compound 8 were evaluated in vitro
using EMT6 and HCT116 cancer cell lines. Cells were treated
with compound 8 at a range of concentrations (0.01–10 mM) and
cultured for 24 h either under normoxia or hypoxia (Oxoid
(
2-nitro-1H-imidazol-1-yl)ethyl)carbamate (8) was synthesised in
three steps and comprised of the essential 2-nitroimidazole
pharmacophore, a short alkyl linker and a TCO moiety for
1
8
2
Anaerogen chamber; 0.1% O ). [ F]FB-Tz was then incubated
with the pre-treated cells for 60 min under normal culture
Scheme 1 Synthesis of (A) tetrazine radiochemistry precursor (3) and reference standard (4) and (B) (E)-cyclooct-3-en-1-yl(2-(2-nitro-1H-
ꢂ
imidazol-1-yl)ethyl)carbamate (8). Reaction conditions: (i) hydrazine hydrate, Ni(OTf)
iii) 4-fluorobenzaldehyde, NaCNBH , RT; (iv) 2-(Boc-amino)ethyl bromide, K CO
2
, MeCN, 60 C, 48 h; (ii) trifluoroacetic acid, DCM, RT, 2 h;
ꢂ
(
3
2
3
, DMF, 60 C, 16 h; (v) trifluoroacetic acid, DCM, RT, 2 h; (vi)
TCO-NHS ester, triethylamine, DMF, RT, 16 h. (C) Radiosynthesis of [ F]FB-Tz; reaction conditions: (vii) [ F]KF, KHCO , K222, MeCN, 90 C,
0 min; (viii) MeCN, 50 C, 20 min followed by flowing the reaction mixture through a custom built BH CN cartridge.
18
18
ꢂ
3
21
ꢂ
ꢀ
1
3
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1
8
in vitro uptake kinetics of [ F]FB-Tz in EMT6 cells incubated
with 10 mM of 8 was determined; between 1 and 2-fold increase
in radioactive uptake was observed every 5 min at the early time
points (5–30 min), which plateaued at 45–60 min, suggesting
rapid in vitro kinetics (Fig. 1C). The toxicity of 8 was evaluated in
vitro using HCT116 and HepG2 cells to determine if pre-
incubation at high concentrations perturbs the biological
system under study. Incubating 8 in concentrations up to 100
mM showed no signicant membrane damage as determined by
LDH activity. The cytotoxicity of 8 incubated in concentrations
up to 100 mM did did not inhibit/minimally inhibited cell
growth in the cell lines over the 72 hour treatment used in the
study. Taken together, compound 8 exhibited low toxicity at
high concentrations (ESI, Fig. S10 and S11†)
1
8
Comparison to a hypoxia radiotracer ([ F]FMISO)
1
8
The uptake of the PET radiopharmaceutical [ F]FMISO was
used as a “gold standard” to demonstrate the detection of
1
8
cellular hypoxia via radioactive uptake assay in live cells. [ F]
FMISO was incubated in both hypoxic/normoxic EMT6 and
HCT116 cell lines for 3 h to compare absolute and relative
uptake with the pretargeted approach. The incubation time of
3
h was selected based on previous studies which showed
18,22–24
optimal uptake at later time-points.
The absolute uptake
1
8
values of [ F]FMISO (3.1 ꢁ 0.4 and 1.3 ꢁ 0.3 %ID per mg for
EMT6 and HCT116, respectively) were considerably lower
compared to the pretargeted approach (90.2 ꢁ 4.2 and 67.7 ꢁ
Fig. 2 (A) Schematic representation of the FASTLab™ cassette for the
18
automated radiosynthesis and purification of [ F]FB-Tz; (B) repre-
18
sentative radio-HPLC chromatograms showing purified [ F]FB-Tz (t
R
2
.9 %ID per mg in EMT6 and HCT116, respectively). The
¼
9:05 mm:ss); (C) representative UV-HPLC chromatogram showing
1
8
an authentic reference standard (t
R
¼ 8:46 mm:ss). There is a 20 s hypoxic : normoxic (H : N) ratio of [ F]FMISO was higher than
delay between the inline UV-detector and g-detector on the analytical for the pretargeted approach (39.3 vs. 12.8 in EMT6 and 20.7 vs.
HPLC system used to run these samples.
3
.5 in HCT116 cells) due to a degree of non-specic uptake of
1
8
the relatively lipophilic [ F]FB-Tz in normoxic cells (<10 %ID
per mg protein). The uptake experiments conrmed that
radioactive accumulation was specic to cellular hypoxia,
however the specicity for detecting hypoxia was dependent
upon higher concentrations (1–10 mM) of 8.
conditions, followed by rigorous washing of the cells with ice
cold PBS before g-counting.
The in vitro uptake (Fig. 3A and B) showed a concentration-
dependent increase in radioactivity accumulation in hypoxic
cells for both EMT6 and HCT116 cell lines with increasing
concentrations of 8 (1–10 mM). While incubation of 8 at
concentrations between 0.001 and 0.1 mM had little impact on
radioactive accumulation in both cell lines, incubation with 1 or
Conclusions
The bioorthogonal IEDDA “click” reaction between a radio-
1
8
labelled cell-permeable reporter molecule ([ F]FB-Tz) and 8 in
live cells was successful and signicantly differentiated hypoxic
and normoxic cells by a 12.8-fold increase in relative uptake.
The use of [ F]FMISO conrmed relevant hypoxia levels in our
model system, evidenced by cellular uptake of 3.1 ꢁ 0.4 %ID
per mg of within 180 min. By pretargeting cellular macromol-
ecules for 24 h under similar hypoxic conditions, we were able
to detect the consequent hypoxic adducts, evidenced by [ F]
FMISO cellular uptake of 90.2 ꢁ 4.2 %ID per mg within 60 min.
Two key differences between the pre-targeting approach and the
direct use of a radiolabelled nitroimidazole are noteworthy.
First, even though the uptake of [ F]FB-Tz was rapid, the pre-
targeted approach leads to a degree of non-specic accumula-
tion of TCO in normoxic cells and consequently [ F]FB-Tz
normoxia labelling, leading to lower hypoxic : normoxic ratio
compared to the [ F]FMISO approach. Second, pretargeting of
1
0 mM led to remarkable uptake; reaching 90.2 ꢁ 4.2 %ID
per mg in EMT6 cells and 67.7 ꢁ 2.9 %ID per mg in HCT116
cells at 10 mM; notably, this was specic to hypoxia, with cells
exhibiting a statistically signicant (P < 0.0001) increase in
radioactive accumulation under hypoxia (12.8-fold in EMT6 and
1
8
3.5-fold in HCT116) compared to normoxia conditions, which
remained consistently low (<10 %ID per mg) at all concentra-
tions of 8 studied. Radioactive accumulation is likely to be
dependent upon the inuence of the intracellular concentration
1
8
1
8
of 8 on IEDDA reaction efficiency, and the molar activity of [ F]
FB-Tz; therefore, radioactive uptake in the lower concentration
range of 8 (0.001–0.1 mM) may improve with high molar activity
1
8
1
8
19
F-tetrazines by reducing competition with non-radioactive F-
1
8
tetrazine equivalent for the limited availability reactive TCO
inside the cell. This was not further explored in this study. The
1
8
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18
Fig. 3 The in vitro uptake of [ F]FB-Tz in (A) EMT6 and (B) HCT116 cells. Cells were treated with 8 (0.01–10 mM) and cultured under normoxia or
1
8
hypoxia (0.1% O
2
) conditions for 24 h prior to radioactive uptake experiment: 0.74 MBq per well of [ F]FB-Tz was added to cells and incubated
18
for 60 min. (C) Time course of [ F]FB-Tz uptake in EMT6 cells in vitro. Cells were treated with 8 (10 mM) for 24 h under hypoxia before incubation
18
18
with 0.74 MBq per well of [ F]FB-Tz for 5, 10, 15, 30, 45 and 60 min. (D) Radioactive uptake of [ F]FMISO (1.48 MBq per well) for 0 min (pulse) or
h under normoxia or hypoxia conditions in EMT6 and HCT116. Data were expressed as a percentage of total radioactivity incorporated into
cells, normalized for total cellular protein. Mean values ꢁ SD (n ¼ 6; *P < 0.05, **P < 0.01 and ****P < 0.0001).
3
1
8
cellular macromolecules combined with the use of [ F]FB-Tz answering biological questions where a sensitive detection
was dependent on drug concentration, and only observed at method is required.
1–10 mM of 8. The lack of toxicity of 8 in the LDH assay (ESI,
Fig. S10†) and ability to achieve similar high concentrations of
Experimental
-nitroimidazoles for imaging by magnetic resonance spec-
2
25
Materials and methods
troscopy support the pretargeting approach. Furthermore, the
physicochemical properties of 8 could be further optimised to
achieve higher hypoxia : normoxia ratios. Beyond the imme-
diate application of this technology to hypoxia measurements,
the higher level of cellular accumulation (ca. 90 %ID per mg vs.
All reagents and solvents were purchased from commercial
sources and were used without further purication unless
otherwise stated. HPLC grade acetonitrile (MeCN), dichloro-
methane (DCM), ethyl acetate (EtOAc), hexane, methanol
3
%ID per mg) could open up possibilities for the effective
(MeOH), triethylamine (TEA), potassium carbonate (K CO ),
2 3
delivery of therapeutic radioisotopes, or drug payloads and
potassium bicarbonate (KHCO ) and triuoroacetic acid (TFA)
were purchased from Sigma Aldrich (Gillingham, Dorset, UK).
3
1
8
subsequent monitoring of IEDDA reaction efficiency with [ F]
FB-Tz uptake acting as a pharmacodynamic marker; however,
the non-specic uptake of cell-permeable tetrazines may be
a potential challenge to overcome in the utility of this strategy.
We studied this approach in the context of cellular hypoxia, but
(E)-cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate (TCO-NHS
1
ester) was purchased from Jena Bioscience (Jena, Germany). H
1
3
and C NMR spectra were obtained using a Bruker 400 MHz
spectrometer operating at room temperature. Chemical shis
1
8
the general mechanism and [ F]FB-Tz could be adapted and
applied to other biological targets where a differential retention
of TCO can be established within a population of cells. The
described approach could be an important platform to
(d) are reported in parts per million (ppm) and residual solvent
peaks have been used as an internal reference. Peak multiplic-
ities have been abbreviated as follows: s (singlet), d (doublet),
dd (double-doublet), m (multiplet). NMR spectra were analysed
using MestReNova v11 (Santiago de Compostela, Spain).
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Accurate mass spectra were obtained via the Imperial College 3.60–3.43 (m, 2H), 2.38–2.17 (m, 3H), 1.94–1.73 (m, 4H), 1.72–
1
8
13
Department of Chemistry Mass Spectrometry service. [ F] 1.36 (m, 3H). C-NMR (101 MHz, chloroform-d) d: 29.7, 31.0,
Fluoride was produced by a GE PETtrace cyclotron by 16 MeV 32.5, 34.2, 38.6, 41.0, 49.4, 81.4, 126.7, 128.5, 133.0, 142.2, 156.3.
1
8
+
irradiation of enriched [ O]H O target, supplied by Alliance ESI-MS (m/z): [M + H] calcd for C H N O , 309.1563; found,
2
14 21 4 4
Medical Radiopharmacy Ltd (Warwick, UK). The automated 309.1567.
1
8
18
radiosynthesis of [ F]FB-Tz and [ F]FMISO was performed
using the GE FASTlab™ automated synthesis module (GE Radiochemistry
Healthcare Life Sciences, Amersham, UK). The radiochemistry
18
[
F]FB-Tz was synthesised using the GE FASTLab™ automated
1
8
precursor (NITTP) for [ F]FMISO was purchased from ABX
GmbH (Radeberg, Germany). Solid phase extraction (SPE)
cartridges were purchased from Waters (Elstree, Hertfordshire,
UK) and used according to the manufacturers recommended
radiosynthesis platform via reductive amination radiochemistry
18
18
between precursor 3 and 4-[ F]uorobenzaldehyde ([ F]FBA)
Scheme 1). We described the automated procedure else-
(
21
18
where; in brief, aqueous [ F]uoride was dried for the radi-
osynthesis
trimethylanilinium triuoromethansulfonate precursor and
used without further purication. Compound 3 was dissolved in
acetonitrile by the addition of triethylamine to quench the TFA-
salt, then added to the vessel containing [ F]FBA. The imine
intermediate was formed at 65 C for 15 min, before the reac-
tion mixture was owed through a cartridge containing solid-
supported cyanoborohydride to reduce the imine to the
guidelines.
The
4-formyl-N,N,N-trimethylanilinium
tri-
18
of
[ F]FBA
from
the
4-formyl-N,N,N-
1
8

uoromethanesulfonate precursor ([ F]FBA precursor) was
26
synthesised following a literature procedure. Radioactive
product identity was determined by RP-HPLC using an Agilent
1200 series instrument connected to a ow-ram detector (Lab-
18
logic, Sheffield, UK). The system was equipped with a Phenom-
ꢂ
˚
enex Gemini 5m C18 110 A (150 ꢃ 4.6 mm) column; the mobile
phase was A: H O (0.1% TFA) and B: MeCN. The gradient was:
2
0
–1 min, 95% A 1–16 min, 5% A 16–17 min, 95% A 17–20 min,
18
secondary amine. [ F]FB-Tz was puried by semi-preparative
HPLC (Shimadzu LC20-AT pump attached to a custom-built
ꢀ1
95% A at 1 mL min . Elution proles were analysed using
Laura soware (Lablogic, Sheffield, UK).
system, equipped with an Agilent Eclipse XDB-C18, 5m (250 ꢃ
9.4 mm) column. The mobile phase was 20% EtOH/80% sodium
ꢀ1
phosphate (58 mM, pH 2.4) at a ow rate of 4 mL min ) and the
desired product was diluted in water (30 mL), trapped on a tC18
SPE cartridge, dried under a ow of nitrogen and eluted in EtOH
for biological use. [ F]FB-Tz was characterised by radio-HPLC
to determine its identity (by comparison to an authentic refer-
ence standard) and purity (Fig. 2B and C).
Synthesis
The general synthesis for compounds used in this work are
shown in Scheme 1. The synthesis of tetrazine 3 (radiochemistry
precursor) and 2-(2-nitro-1H-imidazol-1-yl)ethan-1-amine (7) are
18
21,27
reported elsewhere.
Synthesis of
tetrazin-3-yl)phenyl)methan amine (4). (4-(6-Methyl-1,2,4,5-
tetrazin-3-yl)phenyl)methanaminium triuoroacetate salt
200 mg, 0.66 mmol) and 4-uorobenzalehyde (81 mg, 0.66
N-(4-uorobenzyl)-1-(4-(6-methyl-1,2,4,5-
Cell culture
EMT6 murine breast cancer cells were a kind gi from Prof.
Soa Pascu (University of Bath, UK) and were grown in Way-
mouth medium (Thermo Fisher Scientic); HCT116 human
colorectal cancer cells (ATCC) were grown in Dulbecco Modied
Eagle Medium (DMEM, Sigma-Aldrich). All media were sup-
plemented with 1% L-glutamine and 2% penicillin–strepto-
mycin (Life Technologies). Waymouth medium contained 15%
fetal calf serum, with 10% fetal calf serum added to DMEM. All
(
mmol) was added to dry MeCN followed by the addition of
sodium triacetoxyborohydride (212 mg, 1.00 mmol). The reac-
tion was stirred for 16 h at ambient temperature. Solvent was
removed in vacuo and the residue puried by column chroma-
tography (1 : 1 EtOAc/hexane, silica) to yield the product as
1
a pink solid (71 mg, 35%). H-NMR (400 MHz, chloroform-d)
d 8.54–8.43 (m, 2H), 7.55–7.47 (m, 2H), 7.30–7.22 (m, 2H),
ꢂ
cells were cultured at 37 C in a humidied atmosphere con-
taining 5% CO . All cells were routinely tested for mycoplasma
and typically not passaged for longer than three months.
7
3
1
1
.00–7.22 (m, 2H), 7.00–6.90 (m, 2H), 3.84 (s, 2H), 3.75 (s, 2H),
.02 (s, 3H). C-NMR (400 MHz, chloroform-d) d 21.1, 52.7,
1
3
2
15.4, 128.06, 128.9, 129.8, 130.6, 135.6, 145.1, 160.8, 163.2,
1
9
64.0, 167.2. F-NMR (376 MHz, chloroform-d) d ꢀ115.77. ESI-
1
8
+
In vitro uptake of [ F]FB-Tz
MS (m/z): [M + H] calcd for C H N F, 310.1486; found,
1
7
17 5
4
310.1464.
EMT6 and HCT116 cells were, respectively, plated at 8 ꢃ 10
5
Synthesis of (E)-cyclooct-3-en-1-yl(2-(2-nitro-1H-imidazol-1- and 2.5 ꢃ 10 cells per well in 6-well plates. Aer 24 h, cells were
yl)ethyl)carbamate (8). 2-(2-Nitro-1H-imidazol-1-yl)ethan-1- treated with 8 at indicated concentrations ranging from 0.01 to
amine (7, 35.1 mg, 0.224 mmol) was dissolved in DMF (2 mL). 10 mM and cultured under normoxia or within a hypoxia (Oxoid
2
Triethylamine (31.2 mL, 0.224 mmol) and (E)-cyclooct-4-enyl 2,5- Anaerogen) chamber; 0.1% O for 24 h. Hypoxia was conrmed
dioxo-1-pyrrolidinyl carbonate (TCO-NHS ester, 50 mg, 0.187 through the change of color of the indicator strip (Oxoid Resa-
mmol) were added and stirred at r.t. for 16 h. The crude was zurin, Thermo Fisher Scientic). On the day of the radioactive
1
8
evaporated and puried by column chromatography (100% uptake experiment, fresh media containing 0.74 MBq of [ F]FB-
EtOAc, silica) to yield the product as a yellow powder (30 mg, Tz was added to individual wells (1 mL per well). Cells were
1
ꢂ
5
2
2% yield). H-NMR (400 MHz, chloroform-d) d 7.07–6.91 (m, incubated with the radiotracer for 60 min at 37 C in a humid-
H), 5.70–5.17 (m, 3H), 4.57–4.34 (m, 2H), 4.32–4.17 (m, 1H), ied condition of 5% CO
2
. Cells were then washed three times
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with ice-cold PBS and lysed in RIPA buffer (1 mL per well) for Statistical analysis
1
0 min on ice. The radioactivity of 800 mL lysate from each
Data were analysed using GraphPad Prism v.8.2.1. The results
were expressed as the mean values ꢁ standard deviation (SD).
The ANOVA was used for multiple comparisons and differences
were dened as signicant if P < 0.05.
sample was counted on a WIZARD2 Automatic Gamma
Counter. Data were expressed as a percentage of incubated
radioactive dose (ID), normalized to total cellular protein (%ID
per mg) as measured by bicinchoninic acid (BCA) assay.
Conflicts of interest
1
8
In vitro uptake of [ F]FMISO
EMT6 and HCT116 cells were plated at 5 ꢃ 10 cells per well in
-well plates and allowed to settle for 24 h. On the day of the
radioactive uptake experiment, fresh media containing 1.48
5
There are no conicts to declare.
6
1
8
Acknowledgements
MBq of [ F]FMISO was added to individual wells (1 mL per
well) and incubated for 0 min (pulse) or 3 h under normoxia or
hypoxia conditions (Oxoid Anaerogen chamber, 0.1% O
This work was supported by the Imperial College NIHR
Biomedical Research Centre award (WSCC_P62585) and the
Medical Research Council grant (MC-A652-5PY80). C. C. was
funded by King's and Imperial College London EPSRC Centre
for Doctoral Training in Medical Imaging (EP/L015226/1).
2
) at
ꢂ
37
C in a humidied condition of 5% CO
2
. Hypoxia was
conrmed through the change of color of the indicator strip
Oxoid Resazurin, Thermo Fisher Scientic). Cells were then
washed three times with ice-cold PBS and lysed in RIPA buffer
1 mL per well) for 10 min on ice. The radioactivity of 800 mL
(
(
lysate from each sample was counted on a WIZARD2 Automatic Notes and references
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