Rotaxane Probes for the Detection of Hydrogen Peroxide by 129Xe HyperCEST NMR Spectroscopy

Article abstract of DOI:10.1002/anie.201903045

The development of sensitive and chemically selective MRI contrast agents is imperative for the early detection and diagnosis of many diseases. Conventional responsive contrast agents used in ¹H MRI are impaired by the high abundance of protons in the body. ¹²⁹Xe hyperCEST NMR/MRI comprises a highly sensitive complement to traditional ¹H MRI because of its ability to report specific chemical environments. To date, the scope of responsive ¹²⁹Xe NMR contrast agents lacks breadth in the specific detection of small molecules, which are often important markers of disease. Herein, we report the synthesis and characterization of a rotaxane-based ¹²⁹Xe hyperCEST NMR contrast agent that can be turned on in response to H₂O₂, which is upregulated in several disease states. Added H₂O₂ was detected by ¹²⁹Xe hyperCEST NMR spectroscopy in the low micromolar range, as well as H₂O₂ produced by HEK 293T cells activated with tumor necrosis factor.

Full text of DOI:10.1002/anie.201903045

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Accepted Article
Title: Rotaxane probes for the detection of hydrogen peroxide by
129Xe hyperCEST NMR
Authors: Sarah H. Klass, Ashley E. Truxal, Tahoe A. Fiala, Joseph
Kelly, Dang Nguyen, Joel A. Finbloom, David E. Wemmer,
Alexander Pines, and Matthew B. Francis
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903045
Angew. Chem. 10.1002/ange.201903045
Link to VoR: http://dx.doi.org/10.1002/anie.201903045
http://dx.doi.org/10.1002/ange.201903045

10.1002/anie.201903045
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Rotaxane probes for the detection of hydrogen peroxide by 129Xe
hyperCEST NMR
Sarah H. Klass,a Ashley E. Truxal,a Tahoe A. Fiala,a Joseph Kelly,a Dang Nguyen,a Joel Finbloom,a
David E. Wemmer,a Alexander Pines,a,b Matthew B. Francis a,b,
*
b
aDepartment of Chemistry, University of California, Berkeley, and Materials Sciences Division, Lawrence
Berkeley National Laboratories, Berkeley, California 94720.
*mbfrancis@berkeley.edu
in biological samples, enabling direct and quantitative
imaging of local spin environments that are often
indistinguishable via conventional MRI.7 The combination of
this acute sensitivity and xenon’s high solubility in blood,
tissue, and lipophilic membranes distinguishes it from other
heteronuclear contrast agents.8 Further, facile
Abstract: The development of sensitive and chemically
selective MRI contrast agents is imperative for the early
detection and diagnosis of many diseases. Conventional
1
responsive contrast agents used in H MRI are impaired by
the high abundance of protons in the body. 129Xe hyperCEST
NMR/MRI comprises a highly sensitive complement to
traditional 1H MRI due to its ability to report specific chemical
environments. To date, the scope of responsive 129Xe NMR
contrast agents lacks breadth in the specific detection of small
molecules, which are often important markers of disease.
Herein, we report the synthesis and characterization of a
rotaxane-based 129Xe hyperCEST NMR contrast agent that
can be turned on in response to H2O2, which is upregulated
as a result several disease states. Added H2O2 was detected
by 129Xe hyperCEST NMR in the low µM range, as well as
H2O2 produced by HEK 293T cells activated with tumor
necrosis factor.
hyperpolarization of 129Xe nuclei can be obtained through
spin exchange optical pumping, leading to contrast
enhancements of 4-5 orders of magnitude over thermal
polarization.9 The lack of background 129Xe signals in most
systems gives rise to uncomplicated spectra as well as the
potential for improved resolution of small, localized
populations of target molecules.10
The sensitive and selective detection of disease markers is
of high importance for the identification and treatment of a
variety of human diseases. Magnetic resonance imaging
(MRI), the clinical version of nuclear magnetic resonance
(NMR) spectroscopy, is a noninvasive imaging technique
that can achieve high resolution and excellent tissue
penetration. Conventional MRI techniques rely on differences
in the relaxation properties of protons primarily associated
with water molecules in different tissue types. Typically,
contrast agents in the form of paramagnetic metal chelates
or spin-labeled biomolecules alter the relaxation properties of
local water molecules to provide additional structural and
functional information.1 Responsive 1H and 13C NMR
contrast agents have also been developed that produce a
signal only in the presence of particular disease markers,
such as enzymes, signaling molecules, and oxidizing
conditions.2,3,4,5 This approach can aid in early disease
detection and provides insight into the chemical environment,
ultimately enabling a better understanding of disease
pathology. However, even with these contrast agents, signal
and sensitivity are often lost due to spectral complexity within
the limited chemical shift range that protons possess.
Figure 1. Hydrogen peroxide-sensing rotaxane for 129Xe
hyperCEST NMR. CB6 (orange) is threaded along a
molecular axle (gray) that is mechanically locked into
place by the presence of two bulky stoppers (blue and
green), creating a supramolecular complex known as a
rotaxane. The presence of the axle in the interior of CB6
prevents Xe from entering the cavity and thus presents
no Xe@CB6 signal in the hyperCEST NMR spectrum
(OFF). After cleavage of one of the bulky stoppers by a
reaction with H2O2, the CB6 cavity becomes available to
form a host-guest complex with Xe atoms. Insets: When
inside the CB6 cavity, the Xe experiences a relatively
shielded environment, creating a unique chemical shift
observable by Xe NMR. An RF saturation pulse
corresponding to this chemical shift can be applied to
depolarize the Xe selectively inside the CB6. The
reduction of the bulk hyperpolarized Xe (Xe@H2O) signal
as a result of depolarizing xenon at the Xe@CB6
chemical shift is then observed (ON). Recording the
Xe@H2O signals using an array of saturation frequencies
yields the above z-spectra.
Hyperpolarized xenon chemical exchange saturation transfer
(
129Xe hyperCEST) NMR comprises a sensitive complement
to traditional 1H MR imaging approaches.6 Although it is a
non-toxic and non-reactive noble gas, xenon is very sensitive
to its local chemical environment due to its high electron
polarizability. The easy deformation of its electron cloud
leads to chemical shifts that range over several hundred ppm
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Being chemically inert, 129Xe is effectively functionalized for
selective detection by participating in supramolecular host-
guest interactions that cause either large chemical shifts or
changes in relaxation parameters that are distinct to the host
environments in which 129Xe resides.11,12,13,7 Since the
majority of xenon atoms are present in bulk solution and
undergo continuous exchange with host molecules, we can
exploit the exchange between host-bound and “free” xenon
by way of CEST techniques to attain detection limits of hosts
down to sub-nM levels.14,15
species known as a rotaxane.19,20 By designing one of these
bulky stoppers to be cleaved under specific conditions, such
as activation by a disease marker, a Xe@CB6 signal can be
selectively observed only when activated. Using these
design principles, our group and others have previously
produced rotaxanes and rotaxane-like species where the
CB6 response is occluded by a molecular species threaded
through the cavity. These systems have been designed to
release CB6 under a variety of conditions, such as strong
base,14 cancer-related matrix-metalloproteinase (MMP)
enzymes,21 and the human carbonic anhydrase enzyme.22
This “turn on” effect differs in concept from other systems,
such as the previously reported biotinylated CB7 construct,
which exhibits a unique NMR shift upon binding to its protein
target.23 In other work, cyclodextrin based pseudorotaxanes
have also provided encouraging results.24
To create “turn-on” 129Xe hyperCEST NMR contrast agents,
researchers have used the known Xe host cucurbit[6]uril
(CB6) due to its favorable exchange parameters for
hyperCEST applications.15,16 CB6 is a toroidal molecule that
can harbor a single 129Xe atom in its interior cavity, creating a
distinct 129Xe@CB6 chemical shift.17 CB6 has been used as
an in vivo contrast agent in rat vasculature, highlighting the
applicability of future designs based on this moiety.18 To
prevent 129Xe molecules from entering the interior of CB6
and producing this unique signal until desired, the CB6 can
be threaded onto a molecular axle. This axle is then capped
by two bulky stopper groups, creating the supramolecular
Based on the design elements of these first examples, we
sought to create a probe that would be responsive to
increased levels of hydrogen peroxide (H2O2, Figure 1). Prior
research has implicated upregulated levels of H2O2
production in the advancement of inflammation, and
increases in oxidative stress have been correlated with the
Figure 2. Convergent synthesis of Rotaxane 1 and Rotaxane 2. (a) The synthetic route to produce a H2O2 sensing cap
involved three steps, and (b) the synthetic route to produce a paraxylene diamine axle required four steps. (c) The two
segments were combined using a 2-step procedure and subsequently threaded with CB6. (d) The axles were then capped
with either fluorophore or maleimide functionalities resulting in the formation of Rotaxane 1 and Rotaxane 2 respectively. LC-
MS traces are shown for Rotaxane 1 (expected m/z: 1813.44; observed m/z: 1813.53, boron isotope pattern observed) and
Rotaxane 2 (expected m/z: 1674.37; observed m/z: 1674.56, boron isotope pattern observed).
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10.1002/anie.201903045
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advancement of cancer25, diabetes26, cardiovascular
disease, pulmonary disease, and wound healing.27,28 The
high sensitivity of 129Xe hyperCEST NMR could allow for the
detection of extracellular H2O2 at the low physiological levels
(0.5-50 µM) associated with these disease states.29,27
129Xe MRI has demonstrated a unique strength in functional
lung imaging, making oxidatively sensitive sensors most
provocative for the detection of pulmonary afflictions in which
abnormally increased levels of H2O2 have been linked.
Known examples include chronic obstructive pulmonary
disease, community-acquired pneumonia, cancer of the lung,
and denervation-induced muscle atrophy.18,19,20 Current
examples for imaging H2O2 in vivo or in opaque
environments include chemiluminescent nanoparticles, a
13C-labeled MRI probe, and a bioluminescent reporter.4,33,34
However, these techniques suffer from either limited
sensitivity or the requirement of modifying the natural sample
environment. Therefore, imaging H2O2 in an unperturbed
environment at physiologically relevant concentrations
remains an interesting challenge. Herein we describe the first
synthesis, characterization, and application of a 129Xe
hyperCEST contrast agent for the detection of H2O2.
Several design elements were considered in the synthesis of
a rotaxane that could function as a 129Xe hyperCEST turn-on
probe in response to H2O2: (1) Inspired by the design of
several H2O2 activated fluorescent, positron emission
tomography (PET), and 1H/13C MRI probes, we incorporated
a sensitive and selective aryl boronic acid group as a
cleavable cap on the rotaxane (Figure 2a);34,35,36,37,29 (2) To
improve sensitivity, the rotaxane axle was composed of a p-
xylene diamine moiety that has one of the lowest reported Ka
values for CB6 (5.5 x102 M-1, Figure 2b);19 (3) To allow for
ease of synthesis and modification to the rotaxane design, a
convergent route was designed, in which the axle and cap
were joined at later steps (Figure 2c). Using this route, two
such rotaxanes were prepared with differing non-responsive
capping moieties. Rotaxane 1 was capped with a fluorophore
to allow for specific UV detection during HPLC/LC-MS
cleavage studies (Figure 2d). Rotaxane 2 was capped with a
maleimide functional group that allows for further conjugation
to biomolecules and was used in the 129Xe hyperCEST
experiments (Figure 2e). Both molecules were purified using
RP-HPLC before use.
Figure 3. HPLC and ESI-TOF MS analysis of rotaxane
cleavage by H2O2. (a) The method of cleavage by H2O2
is shown for Rotaxane 1. (b) HPLC analyses followed
the absorbance over the course of 16 h. A control
sample with no H2O2 added was used to determine if
any degradation of the rotaxane occurred. Starting
Rotaxane 1 and oxidized Rotaxane 3 both eluted at 14
min, but could be distinguished using ESI-TOF MS. The
peak that emerges at 13 min was identified as cleaved
pseudo-rotaxane product 4. (c) Integration of the peak
area at 13 min afforded a linear relationship with respect
to time. (d) MS analysis of peak at 13 min is shown:
expected m/z: 1534.43; observed m/z: 1534.48. MS
analysis of peak at 14 min corresponded to the intact
rotaxane, with the boronic acid group present only in
samples that had not been exposed to H2O2 (0 and 16 h
time points). In all samples that had been exposed to
H2O2 the MS trace corresponded solely to the oxidized
phenol rotaxane (expected m/z: 1785.62; observed m/z:
1786.52).
To determine the cleavage rates of the rotaxane caps in the
presence of excess H2O2, a sample of Rotaxane 1 was
monitored over time by HPLC/LC-MS. Rotaxane 1 was found
to be completely oxidized within 1 h. However, the 1,6
elimination reaction that ultimately results in bond cleavage
proved to be the slow step, with less than 50% cleavage
after 16 h. Kinetic measurements extrapolated from these
data (assuming pseudo-first-order conditions with 7.5 µM
Rotaxane 1 and 100 µM H2O2 in 1xPBS buffer at pH 7.4)
gave a rate constant of k= 8.52 x 10-3 s-1 (Figure 3).
To determine if the sucessful cleavage reaction observed
using LC-MS resulted in a 129Xe hyperCEST signal, multiple
concentrations of Rotaxane 2 and H2O2 were analyzed to
monitor the emergence of the Xe@CB6 signal over time. A
Xe@CB6 hyperCEST response evolved within 1 h following
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10.1002/anie.201903045
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the addition of 2 equivalents of hydrogen peroxide (25 µM
Rotaxane 2, 50 µM H2O2) , and quickly reached and
maintained an apparent maximum saturation of about 25%
(Figure 4a). Previous rotaxane-based sensors have required
8-24 h to demonstrate the same magnitude of CEST
response, demonstrating the notable improvement in
efficiency and sensitivity of this system.14,21 Evaluation of
Rotaxane 2 (Figure 4b) and H2O2 at varying concentrations
(Figure 4c) led to detection limits of 2.5 µM and 5 µM,
respectively. Notably, the limits of detection could be
significantly improved by new advancements in
acquisition of a Xe@CB6 response indicates that enough
CB6 is released for xenon exchange into and out of the
cavity.
The maleimide cap on Rotaxane 2 enabled the
functionalization of cysteine residues present on the vascular
cell adhesion molecule (VCAM)-1 binding peptide and a
tobacco mosaic virus (TMV) protein-based nanoparticle. The
VCAM-1 binding peptide was chosen because this receptor
has implications in inflammatory disease and cancer.42,43
TMV is a disk shaped nanoparticle that assembles into a
nanoscale structure that is 17 nm in diameter and composed
of 34 protein monomers.44 This protein assembly was
chosen for is distinct morphology, which could be
hyperpolarization methods, where 129Xe hyperpolarization
levels of greater than 50% have been obtained.38 This is a
large enhancement over the polarization levels used in this
study, which were only 0.05-2%.
adventageous in drug delivery and imaging applications.45,46
Using excess Rotaxane 2, the VCAM-1 peptide was
completly modified, while the TMV nanoparticle was
purposefully modified at 30% of its available sites, resulting
in 10 rotaxane molecules per TMV disk (Figure S1).
We then sought to detect endogenously produced H2O2 by
exposing HEK 293T cells to tumor necrosis factor alpha
(TNF), a signaling protein involved in immune cell regulation
that causes an increase in cellular H2O2 production.47,48
Adherent cells in 6 well plates were either exposed to 40 µg
of TNF (TNF+) or remained untreated (TNF–). Both samples
were incubated for 6 h at 37 °C under an atmosphere of 5%
Figure 4. Rotaxane 2 acts as a 129Xe hyperCEST NMR
probe for H2O2. (a)The signal at 20 kHz corresponds to
bulk dissolved xenon in water and is always present, but
upon addition of H2O2, cleavage of Rotaxane 2 releases
CB6. This leads to the immediate appearance of a
Xe@CB6 CEST response at 12 kHz that builds over
time. (b,c) The signal can be observed down to 5 µM
Rotaxane 2 and 2.5 µM H2O2.
Although the observed response magnitudes did increase
with increasing Rotaxane 2 or H2O2 concentration, they did
not seem to be able to achieve more than 25-28% saturation.
In contrast, a 5 µM solution of free CB6 that was not
previously threaded onto a rotaxane provided nearly
complete saturation immediately using the same acquisition
parameters. One explanation for the incomplete saturation
could be the formation of a complex between the carbonyl-
rich portal of CB6 and the protonated amine remaining at the
end of the cleaved rotaxane. CB6 is known to form such
complexes with short peptides as well as long diammonium
alkanes.39,40,41 This includes the p-xylylene diamine
group used in this study.19 Indeed, we can observe an
intact CB6•4 complex by HPLC/LCMS after axle cleavage by
H2O2. Furthermore, the mass spectra indicate that H2O2
does not affect the molecular structure of the CB6 or the
cleaved axle. Presumably the interaction with the cleaved
axle sequesters some of the CB6, thus limiting the saturation
level that is observed. Nonetheless, the successful
Figure 5. Detection of a 129Xe hyperCEST signal in
response to endogenously produced H2O2. TMV-
Rotaxane 2 was added to the supernatants from HEK
293T cells with (TNF+) or lacking (TNF–) TNF in the
cultures. The samples were monitored for a Xe@CB6
CEST response over 24 h. No Xe@CB6 response
appeared for the any of the TNF(–) samples, while a
signal at 10-13 kHz appeared and stabilized within a few
hours for the TNF+ samples.
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10.1002/anie.201903045
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CO2. The supernatant was then treated with the TMV-
Rotaxane 2 conjugate to a final concentration of 1 µM
(corresponding to 10 µM Rotaxane 2 groups). The samples
were examined by 129Xe hyperCEST NMR over the course of
several hours (Figure 5). No Xe@CB6 signal evolved for the
TNF– sample, even after 8 h. In contrast, a response
evolved and stabilized after 2 h for the TNF+ sample. The
Xe@CB6 signal, albeit significantly broadened, persisted
even after 24 h. By comparison with the experiment in which
exogenous H2O2 was added to HEK 293T cells, we estimate
low µM H2O2 was expressed as a result of incubation with
TNF. This is well within the detection limit of both sensor and
target, demonstrating the utility of this technique to detect
biologically relevant concentrations.
Caspase-Responsive Smart Gadolinium-Based
Contrast Agent for Magnetic Resonance Imaging of
Drug-Induced Apoptosis. Chem. Sci. 2014, 5 (10),
3845.
(4)
(5)
Lippert, A. R.; Keshari, K. R.; Kurhanewicz, J.;
Chang, C. J. A Hydrogen Peroxide-Responsive
Hyperpolarized 13 C MRI Contrast Agent. J. Am.
Chem. Soc. 2011, 133 (11), 3776–3779.
Angelovski, G.; Gottschalk, S.; Milošević, M.;
Engelmann, J.; Hagberg, G. E.; Kadjane, P.; Andjus,
P.; Logothetis, N. K. Investigation of a Calcium-
Responsive Contrast Agent in Cellular Model
Systems: Feasibility for Use as a Smart Molecular
Probe in Functional MRI. ACS Chem. Neurosci.
2014, 5 (5), 360–369.
Witte, C.; Kunth, M.; Döpfert, J.; Rossella, F.;
Schröder, L. Hyperpolarized Xenon for NMR and
MRI Applications. J. Vis. Exp. 2012, No. 67.
Wang, Y.; Dmochowski, I. J. An Expanded Palette of
Xenon-129 NMR Biosensors. Acc. Chem. Res. 2016,
49 (10), 2179–2187.
In conclusion, we have demonstrated the synthesis of a novel
CB6 rotaxane that enables the selective detection of
endogenously produced H2O2 by 129Xe hyperCEST NMR.
These results provide an important foundation for future
applications focusing on the diagnostic imaging of diseased
tissue in which peroxide levels are abnormally increased.
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(7)
Experimental Section
(8)
(9)
Mcmahan, Z. H.; Wigley, F. M. HHS Public Access;
2015; Vol. 5.
Walker, T. G.; Happer, W. Spin-Exchange Optical
Pumping of Noble-Gas Nuclei. Rev. Mod. Phys.
1997, 69 (2), 629–642.
Oros, A. M.; Shah, N. J. Hyperpolarized Xenon in
NMR and MRI. Phys. Med. Biol. 2004, 49 (20).
Bartik, K.; Luhmer, M.; Dutasta, J.-P.; Collet, A.;
Reisse, J. 129 Xe and 1 H NMR Study of the
Reversible Trapping of Xenon by Cryptophane-A in
Organic Solution. J. Am. Chem. Soc. 1998, 120 (4),
784–791.
Spence, M. M.; Rubin, S. M.; Dimitrov, I. E.; Ruiz, E.
J.; Wemmer, D. E.; Pines, A.; Yao, S. Q.; Tian, F.;
Schultz, P. G. Functionalized Xenon as a Biosensor.
Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (19), 10654–
10657.
Palaniappan, K. K.; Ramirez, R. M.; Bajaj, V. S.;
Wemmer, D. E.; Pines, A.; Francis, M. B. Molecular
Imaging of Cancer Cells Using a Bacteriophage-
Based (129) Xe NMR Biosensor. Angew. Chem. Int.
Ed. Engl. 2013, 112, 1–6.
Slack, C. C.; Finbloom, J. A.; Jeong, K.; Bruns, C. J.;
Wemmer, D. E.; Pines, A.; Francis, M. B. Rotaxane
Probes for Protease Detection by 129 Xe HyperCEST
NMR. Chem. Commun. 2017, 53 (6), 1076–1079.
Wang, Y.; Dmochowski, I. J. Cucurbit[6]Uril Is an
Ultrasensitive 129 Xe NMR Contrast Agent. Chem.
Commun. 2015, 51 (43), 8982–8985.
Full experimental methods can be found in the Supporting
Information.
Acknowledgements
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This work was supported by the UCSF Hana Jabsheh Fund.
S.H.K. was supported by the Chemical Biology Graduate
Program at UC Berkeley (NIH T32-GM066698). J.A.F. was
supported under contract FA9550-11-C-0028 and awarded
by the Department of Defense, Air Force Office of Scientific
Research, National Defense Science and Engineering
Graduate (NDSEG) Fellowship, 32 CFR 168a.
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(13)
(14)
Keywords:
Rotaxanes
129Xe hyperCEST NMR
NMR spectroscopy
CB6: Cucurbit[6]uril
References
(1)
Caravan, P. Strategies for Increasing the Sensitivity
of Gadolinium Based MRI Contrast Agents. Chem.
Soc. Rev. 2006, 35 (6), 512.
(2)
Park, J. Y.; Baek, M. J.; Choi, E. S.; Woo, S.; Kim, J.
H.; Kim, T. J.; Jung, J. C.; Chae, K. S.; Chang, Y.;
Lee, G. H. Paramagnetic Ultrasmall Gadolinium
Oxide Nanoparticles as Advanced T 1 MRI Contrast
Agent: Account for Large Longitudinal Relaxivity,
Optimal Particle Diameter, and In Vivo T 1 MR
Images. ACS Nano 2009, 3 (11), 3663–3669.
Ye, D.; Shuhendler, A. J.; Pandit, P.; Brewer, K. D.;
Tee, S. S.; Cui, L.; Tikhomirov, G.; Rutt, B.; Rao, J.
(15)
(16)
Barrow, S. J.; Kasera, S.; Rowland, M. J.; del Barrio,
J.; Scherman, O. A. Cucurbituril-Based Molecular
Recognition. Chem. Rev. 2015, 115 (22), 12320–
12406.
(3)
(17)
Kim, B. S.; Ko, Y. H.; Kim, Y.; Lee, H. J.;
This article is protected by copyright. All rights reserved.

10.1002/anie.201903045
Angewandte Chemie International Edition
COMMUNICATION
Selvapalam, N.; Lee, H. C.; Kim, K. Water Soluble
Cucurbit[6]Uril Derivative as a Potential Xe Carrier
for 129Xe NMR-Based Biosensors. Chem. Commun.
2008, 0 (24), 2756.
Hane, F. T.; Li, T.; Smylie, P.; Pellizzari, R. M.; Plata,
J. A.; DeBoef, B.; Albert, M. S. In Vivo Detection of
Cucurbit[6]Uril, a Hyperpolarized Xenon Contrast
Agent for a Xenon Magnetic Resonance Imaging
Biosensor. Sci. Rep. 2017, 7 (1), 41027.
Bruns, C. J.; Stoddart, J. F. The Nature of the
Mechanical Bondꢀ: From Molecules to Machines.
Bruns, C. J.; Stoddart, J. F. Rotaxane-Based
Molecular Muscles. Acc. Chem. Res. 2014, 47 (7),
2186–2199.
Finbloom, J. A.; Slack, C. C.; Bruns, C. J.; Jeong, K.;
Wemmer, D. E.; Pines, A.; Francis, M. B.; Shen, C.;
New, E. J.; Major, J. L.; et al. Rotaxane-Mediated
Suppression and Activation of Cucurbit[6]Uril for
Molecular Detection by 129 Xe HyperCEST NMR.
Chem. Commun. 2016, 52 (15), 3119–3122.
Wang, Y.; Roose, B. W.; Philbin, J. P.; Doman, J. L.;
Dmochowski, I. J. Programming A Molecular Relay
for Ultrasensitive Biodetection through (129) Xe
NMR. Angew. Chem. Int. Ed. Engl. 2016, 55 (5),
1733–1736.
Soc. 2014, 136 (3), 874–877.
(30)
Nowak, D.; Kasielski, M.; Antczak, A.; Pietras, T.;
Bialasiewicz, P. Increased Content of Thiobarbituirc
Acid Reactive Substances and Hydrogen Peroxide in
the Expired Breath Condensate of Patients with
Stable Chronic Obstructive Pulmonary Disease: No
Significant Effect of Cigarette Smoking. Respir. Med.
1999, 93 (6), 389–396.
Majewska, E.; Kasielski, M.; Luczynski, R.; Bartosz,
G.; Bialasiewicz, P.; Nowak, D. Elevated Exhalation
of Hydrogen Peroxide and Thiobarbituric Acid
Reactive Substances in Patients with Community
Acquired Pneumonia. Respir. Med. 2004, 98 (7),
669–676.
Muller, F. L.; Song, W.; Jang, Y. C.; Liu, Y.; Sabia,
M.; Richardson, A.; Van Remmen, H. Denervation-
Induced Skeletal Muscle Atrophy Is Associated with
Increased Mitochondrial ROS Production. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 2007, 293 (3),
R1159–R1168.
Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari,
M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N. In
Vivo Imaging of Hydrogen Peroxide with
Chemiluminescent Nanoparticles. Nat. Mater. 2007,
6 (10), 765–769.
Van de Bittner, G. C.; Dubikovskaya, E. A.; Bertozzi,
C. R.; Chang, C. J. In Vivo Imaging of Hydrogen
Peroxide Production in a Murine Tumor Model with a
Chemoselective Bioluminescent Reporter. Proc.
Natl. Acad. Sci. U. S. A. 2010, 107 (50), 21316–
21321.
Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C.
J. An ICT-Based Approach to Ratiometric
Fluorescence Imaging of Hydrogen Peroxide
Produced in Living Cells. J. Am. Chem. Soc. 2008,
130 (14), 4596–4597.
(18)
(31)
(32)
(19)
(20)
(21)
(22)
(23)
(24)
(33)
(34)
Truxal, A. E.; Cao, L.; Isaacs, L.; Wemmer, D. E.;
Pines, A. Directly Functionalized Cucurbit[7]Uril as a
Biosensor for the Selective Detection of Protein
Interactions by 129 Xe HyperCEST NMR. Chem. – A
Eur. J. 2019, chem.201900610.
Hane, F. T.; Fernando, A.; Prete, B. R. J.; Peloquin,
B.; Karas, S.; Chaudhuri, S.; Chahal, S.;
(35)
(36)
Shepelytskyi, Y.; Wade, A.; Li, T.; et al. Cyclodextrin-
Based Pseudorotaxanes: Easily Conjugatable
Scaffolds for Synthesizing Hyperpolarized Xenon-
129 Magnetic Resonance Imaging Agents. ACS
Omega 2018, 3 (1), 677–681.
Szatrowski, T. P.; Nathan, C. F. Production of Large
Amounts of Hydrogen Peroxide by Human Tumor
Cells. Cancer Res. 1991, 51 (3), 794–798.
Houstis, N.; Rosen, E. D.; Lander, E. S. Reactive
Oxygen Species Have a Causal Role in Multiple
Forms of Insulin Resistance. Nature 2006, 440
(7086), 944–948.
Niethammer, P.; Grabher, C.; Look, A. T.; Mitchison,
T. J. A Tissue-Scale Gradient of Hydrogen Peroxide
Mediates Rapid Wound Detection in Zebrafish.
Nature 2009, 459 (7249), 996–999.
Reuter, S.; Gupta, S. C.; Chaturvedi, M. M.;
Aggarwal, B. B. Oxidative Stress, Inflammation, and
Cancer: How Are They Linked? Free Radic. Biol.
Med. 2010, 49 (11), 1603–1616.
Carroll, V.; Michel, B. W.; Blecha, J.; VanBrocklin,
H.; Keshari, K.; Wilson, D.; Chang, C. J. A Boronate-
(25)
(26)
18 F]FLT Probe for Hydrogen Peroxide
Caged [
Detection Using Positron Emission Tomography. J.
Am. Chem. Soc. 2014, 136 (42), 14742–14745.
Lin, V. S.; Dickinson, B. C.; Chang, C. J. Boronate-
Based Fluorescent Probes: Imaging Hydrogen
Peroxide in Living Systems. Methods Enzymol.
2013, 526.
Nikolaou, P.; Coffey, A. M.; Walkup, L. L.; Gust, B.
M.; Whiting, N.; Newton, H.; Barcus, S.; Muradyan,
I.; Dabaghyan, M.; Moroz, G. D.; et al. Near-Unity
Nuclear Polarization with an Open-Source 129Xe
Hyperpolarizer for NMR and MRI. Proc. Natl. Acad.
Sci. 2013, 110 (35), 14150 LP-14155.
(37)
(38)
(27)
(28)
(29)
(39)
Meschke, C.; Buschmann, H. J.; Schollmeyer, E.
Complexes of Cucurbituril with Alkyl Mono- and
Diammonium Ions in Aqueous Formic Acid Studied
by Calorimetric Titrations. Thermochim. Acta 1997,
Weinstain, R.; Savariar, E. N.; Felsen, C. N.; Tsien,
R. Y. In Vivo Targeting of Hydrogen Peroxide by
Activatable Cell-Penetrating Peptides. J. Am. Chem.
This article is protected by copyright. All rights reserved.

10.1002/anie.201903045
Angewandte Chemie International Edition
COMMUNICATION
297 (1–2), 43–48.
(40)
Buschmann, H. J.; Mutihac, L.; Mutihac, R. C.;
Schollmeyer, E. Complexation Behavior of
Cucurbit[6]Uril with Short Polypeptides. Thermochim.
Acta 2005, 430 (1–2), 79–82.
(41)
Danylyuk, O.; Fedin, V. P.; Sashuk, V. Kinetic
Trapping of the Host-Guest Association Intermediate
and Its Transformation into a Thermodynamic
Inclusion Complex. Chem. Commun. 2013, 49 (18),
1859–1861.
(42)
Chen, Y.; Molnár, M.; Li, L.; Friberg, P.; Gan, L.-M.;
Brismar, H.; Fu, Y. Characterization of VCAM-1-
Binding Peptide-Functionalized Quantum Dots for
Molecular Imaging of Inflamed Endothelium. PLoS
One 2013, 8 (12), e83805.
(43)
(44)
Schlesinger, M.; Bendas, G. Vascular Cell Adhesion
Molecule-1 (VCAM-1)-An Increasing Insight into Its
Role in Tumorigenicity and Metastasis. Int. J. Cancer
2015, 136 (11), 2504–2514.
Finbloom, J. A.; Han, K.; Aanei, I. L.; Hartman, E. C.;
Finley, D. T.; Dedeo, M. T.; Fishman, M.; Downing,
K. H.; Francis, M. B. Stable Disk Assemblies of a
Tobacco Mosaic Virus Mutant as Nanoscale
Scaffolds for Applications in Drug Delivery.
Bioconjug. Chem. 2016, 27 (10), 2480–2485.
Finbloom, J.; Aanei, I.; Bernard, J.; Klass, S.;
Elledge, S.; Han, K.; Ozawa, T.; Nicolaides, T.;
Berger, M.; Francis, M. Evaluation of Three
Morphologically Distinct Virus-Like Particles as
Nanocarriers for Convection-Enhanced Drug
Delivery to Glioblastoma. Nanomaterials 2018, 8
(12), 1007.
(45)
(46)
Adriani, G.; de Tullio, M. D.; Ferrari, M.; Hussain, F.;
Pascazio, G.; Liu, X.; Decuzzi, P. The Preferential
Targeting of the Diseased Microvasculature by Disk-
like Particles. Biomaterials 2012, 33 (22), 5504–
5513.
(47)
(48)
Dixon, S. J.; Stockwell, B. R. The Role of Iron and
Reactive Oxygen Species in Cell Death. Nat. Chem.
Biol. 2014, 10 (1), 9–17.
Martinez-Outschoorn, U. E.; Lin, Z.; Trimmer, C.;
Flomenberg, N.; Wang, C.; Pavlides, S.; Pestell, R.
G.; Howell, A.; Sotgia, F.; Lisanti, M. P. Cancer Cells
Metabolically "Fertilize" the Tumor
Microenvironment with Hydrogen Peroxide, Driving
the Warburg Effect: Implications for PET Imaging of
Human Tumors. Cell Cycle 2011, 10 (15), 2504–
2520.
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Entry for the Table of Contents
COMMUNICATION
The CB6 molecule can host single
129Xe atoms in its interior cavity,
creating a distinct chemical
Sarah H. Klass, Ashley E. Truxal,
Tahoe A. Fiala, Joseph Kelly, Dang
Nguyen, Joel A. Finbloom, David E.
Wemmer, Alexander Pines, Matthew
B. Francis
environment that can be observed
by 129Xe hyperCEST NMR. To
create a “turn on” probe, CB6 can
be threaded onto a molecular axle
and capped by two stoppers to
prevent the entry of 129Xe into the
CB6 interior. Selective cleavage of
one stopper group by H2O2
Page No. – Page No.
Rotaxane probes for the detection
of hydrogen peroxide by 129Xe
hyperCEST NMR
releases CB6, producing a
hyperCEST response.
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