.
Angewandte
Communications
we introduce a new approach for the development of activity-
based probes compatible with MC technology using a compact
telluroether scaffold.
hypoxic environment. This produces protein-2-(amino)imi-
dazole adducts (7) effectively “tagging” the cell. Generation
of the initial nitro radical (4) is a rapidly reversible process in
the presence of molecular oxygen and thus generation of the
active nitrenium labeling agent is slow under normoxic
conditions. In the case of Pimo and EF-5, the detection is
mediated by immunostaining, although similar 2-NI com-
Tellurium provides favorable characteristics for the devel-
opment of small MC probes. The element forms telluroether
and tellurophene functionalities which are small and lipo-
philic; properties which we hypothesize will minimize pertur-
bation of attached biologically sensitive functionalities (i.e.
activity-based groups), thus preserving their biological activ-
ity. This is in direct contrast to the lanthanide-chelating
polymers used as mass tags in MaxPar reagents, because these
groups are large and polar (Figure 1a). Additionally, five
stable tellurium isotopes are available commercially, thereby
allowing for multiple MC-distinguishable probes to be
generated using identical chemistry. As a proof of concept
of such a probe, we have targeted cellular hypoxia due to its
importance in tumor biology and the well-defined chemical
methods for interrogation.
1
8
pounds have been developed which incorporate F to allow
[
12]
for detection through positron emission tomography.
Recently, hypoxia probes based on 2-NIs have been devel-
oped that implement alternative imaging modalities such as
single-photon emission computed tomography, magnetic
[13]
resonance imaging, or near-infrared fluorescence.
To adapt the 2-NI functionality for MC detection we
identified an organotellurium scaffold that was compact,
amenable to high yielding synthesis, stable under physiolog-
ical conditions, and relatively nontoxic. Reports of organo-
tellurium toxicity have focused mainly on aryltelluroethers;
however, the limited reports available suggest the more
The development of hypoxic regions is a common char-
acteristic of most solid tumors and is associated with radiation
and chemotherapy resistance, as well as increased metasta-
[15–17]
compact dialkyl telluroethers are less toxic.
Focusing on
telluroethers, we opted to use a compact unsymmetric methyl
alkyl telluride functionality (1, Scheme 1a) with a reactive
hydroxy handle for further chemical manipulation. This group
was accessed through treatment of elemental tellurium with
methyllithium, followed by alkylation of the resultant nucle-
[
8]
sis. Levels of hypoxia also vary widely amongst patients, and
are strongly associated with poor clinical outcome in several
tumor types. Within individual tumors, hypoxia is spatially
heterogeneous and often characterized by gradients of oxy-
genation extending from normal levels near the vasculature to
ophilic methyltelluride salt by 3-chloropropan-1-ol (Sche-
[
9]
[18]
near anoxia at the borders with necrosis. Importantly,
oxygen levels in some tumor regions also fluctuate over
time due to transient changes in vessel perfusion. Areas of
fluctuating hypoxia may be particularly important in driving
poor prognosis, but at present are difficult to quantitate in
me 1a).
The resultant telluroether alcohol (1) was then
treated with p-nitrophenyl chloroformate to afford the
carbamylating reagent 2. The primary amine-bearing 2-NI
scaffold (3) was then easily carbamylated to afford the final
hypoxia probe, designated “Telox” (Scheme 1a). This com-
pound is stable for months if stored in the dark as a solid and
under an inert atmosphere at À208C. At ambient light and
[10]
patients. Current understanding of tumor hypoxia has been
greatly facilitated by the availability of chemical probes
constructed around a 2-nitroimidazole (2-NI) functionality
oxygen levels the probe has a t1 > 48 h in solution (see the
/2
[
11]
such as pimonidazole (Pimo) and EF-5. These probes are
reduced by one electron reductases, such as PORs (p450
oxidoreductase), which act on a variety of nitroaryl com-
Supporting Information). Due to a downfield shift of the Te-
1
CH resonance in the H NMR spectrum of the probe after
3
prolonged exposure to ambient atmosphere, we hypothesize
that the major degradation product upon exposure to
atmospheric oxygen is the telluroxide species or a hydrate
thereof.
[
14]
pounds (Scheme 1b). Studies suggest that upon reduction
of the 2-NI to a 2-(hydroxy amino)imidazole (5), hydroxide is
liberated and an electrophilic 2-(nitrenium)imidazole ion (6)
is generated, which reacts irreversibly with free thiols in the
The proliferative toxicity of Telox was measured in
HCT116 cells by confluency analy-
sis (Figure S1). This experiment
suggested that proliferation was
only mildly affected up to the
maximum probe concentration
evaluated (400 mm) under both
normoxic (21% O ) and hypoxic
2
(
0.2% O ) conditions. An orthogo-
2
nal assay was performed using the
reduction of WST-1 as a metric for
metabolic toxicity induced by the
presence of Telox. In Jurkat cells,
this experiment indicated a meta-
bolic IC50 of 200 Æ 20 mm (Fig-
ure S2).
As a surrogate for a POR
enzyme, the sensitivity of Telox to
enzyme-mediated reduction was
Scheme 1. a) a: MeLi (ꢀ1 equiv), THF, 228C, 10 min; b: 3-chloro-1-propanol (1.0 equiv), THF,
À1928C, 2 h, 72%; c: p-nitrophenyl chloroformate (1.05 equiv), pyridine (2.1 equiv), THF, 228C, 2 h,
7
5%; d: methylbromoacetate (1.0 equiv), K CO (1.5 equiv), tetrabutylammonium iodide
2 3
(
0.022 equiv), MeCN, reflux, 3 h, 60%; e: ethylenediamine (4.0 equiv), MeOH, 228C, 18 h, quant.; f:
pyridine (3.0 equiv), MeOH, 228C, 2 h, 70%. b) Enzyme-catalyzed reduction of the 2-nitroimidazole
functionality to produce the electrophilic protein-labeling nitrenium ion 6.
1
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 11473 –11477