A Noncovalent Fluorescence Turn-on Strategy for Hypoxia Imaging

Article abstract of DOI:10.1002/anie.201813397

Hypoxia plays crucial roles in many diseases and is a central target for them. Present hypoxia imaging is restricted to the covalent approach, which needs tedious synthesis. In this work, a new supramolecular host–guest approach, based on the complexation

Full text of DOI:10.1002/anie.201813397

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International Edition: DOI: 10.1002/anie.201813397
German Edition: DOI: 10.1002/ange.201813397
Imaging Agents
A Noncovalent Fluorescence Turn-on Strategy for Hypoxia Imaging
Wen-Chao Geng, Shaorui Jia, Zhe Zheng, Zhihao Li, Dan Ding, and Dong-Sheng Guo*
Dedicated to Professor Yu Liu on the occasion of his 65th birthday
Abstract: Hypoxia plays crucial roles in many diseases and is
a central target for them. Present hypoxia imaging is restricted
to the covalent approach, which needs tedious synthesis. In this
work, a new supramolecular host–guest approach, based on the
complexation of a hypoxia-responsive macrocycle with a com-
mercial dye, is proposed. To exemplify the strategy, a carboxyl-
modified azocalix[4]arene (CAC4A) was designed that binds
to rhodamine 123 (Rho123) and quenches its fluorescence. The
azo groups of CAC4A were selectively reduced under hypoxia,
leading to the release of Rho123 and recovery of its fluores-
cence. The noncovalent strategy was validated through hypoxia
imaging in living cells treated with the CAC4A–Rho123
reporter pair.
bond, was found to break in a reductive microenvironment
and be reduced to aniline derivatives, with the actual chemical
response being a function of oxygen partial pressure.[11] In the
case of the azo group, little fluorescence is typically seen since
the excited energy state is rapidly dissipated due to conforma-
tional changes around the azo bond.[12] This deactivation
=
pathway is eliminated after reduction of the N N double
bond, thus leading to an increase in the fluorescence emission
intensity.[13]
To the best of our knowledge, all reported efforts to
implement hypoxia imaging through use of azo reduction
have been based on a covalent strategy that involves linking
an azo group to a dye (Scheme 1a). However, direct covalent
H
ypoxia is a common feature in most of solid tumors that is
ascribed to the imbalance between the increased oxygen
consumption caused by rapid proliferation and an inadequate
oxygen supply resulting from an altered tumor vasculature.[1]
Hypoxia is correlated with increased metastasis[2] and
increased resistance to radiotherapy, chemotherapy,[3] and
photodynamic therapy.[4] It is also an indicator for tumor
aggression[5] and an indicator of poor prognoses and ther-
apeutic outcomes.[2] Accurate hypoxia detection and imaging
could be helpful in terms of identifying hypoxic-tumor-
bearing patients and formulating suitable clinical treatment
plans.[6]
The low oxygen levels result in the imbalance of cellular
redox states, causing an enhanced reductive stress.[7] The
reduction potential and the activity of various bioreductive
enzymes in hypoxic tumors and that in normoxic tissues are
significantly different.[7,8] Taking advantage of the higher
reductive stress, a variety of hypoxia-selective probes and
drugs have been developed.[9] Most effort associated with
hypoxia imaging and therapy has focused on the nitro group,
which can be reduced to amine by nitroreductase.[10] Alter-
natively, another kind of reducible chemical group, the azo
Scheme 1. Schematic illustration of the design principles of the
conventional covalent (a) and the proposed noncovalent (b) strategies
for creating hypoxia-responsive systems.
attachment approaches, although effective in terms of creat-
ing hypoxia-responsive systems, sometimes suffer from some
disadvantages, including complicated molecular design, time-
consuming and expensive synthesis, activity changes due to
the covalent linker, and potential toxicity enhancement
because of introducing an exogenous moiety on dyes.[11]
Moreover, each azo conjugate was only implemented for
imaging or treating hypoxia by the linked agents.[11a,12]
Supramolecules represent an alternative approach to produce
stimuli-responsive biomedical systems that are more easily
operated and universal.[14] With this regard, we are working to
develop a noncovalent strategy based on the host–guest
interaction between azocalixarene and dye (Scheme 1b). In
this strategy, a dye is pre-included into the cavity of an
[*] W.-C. Geng, Z. Zheng, Z. Li, Prof. D.-S. Guo
College of Chemistry, Key Laboratory of Functional Polymer Materials
(Ministry of Education), State Key Laboratory of Elemento-Organic
Chemistry, Nankai University
Tianjin 300071 (China)
E-mail: dshguo@nankai.edu.cn
S. Jia, Prof. D. Ding
Key Laboratory of Bioactive Materials, Ministry of Education, College
of Life Sciences, Nankai University
Tianjin 300071 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201813397.
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azocalixarene. This supramolecular complexation leads to
a quenching of the fluorescence intensity. Upon encountering
a hypoxic environment, the azo groups of the azocalixarene
get reduced. This leads to the release of the dye and the
recovery of the fluorescence intensity.
To achieve the imaging of hypoxia via such a noncovalent
way, a tailored host–guest reporter pair is needed. The host is
expected to 1) bind strongly towards commercial imaging
dyes to avoid undesired off-target leaking,[14b] 2) “super”-
quench the fluorescence to minimize the imaging back-
ground,[15] 3) most importantly, be hypoxia-responsive to
release the entrapped dye to allow fluorescence recovery.
Accordingly, we designed the artificial receptor 5,11,17,23-
tetrakis[(p-carboxyphenyl)azo]-25,26,27,28-tetra-hydroxy
calix[4]arene (CAC4A) (Figure 1a). Calixarenes were
selected as the macrocyclic scaffold, which have been
fluorescence intensity of CAC4A–Rho123 were observed in
the presence of these biological species. As a result, the
extremely strong binding between CAC4A and Rho123
resists the interference from biologically coexisting species.
The complexation between Rho123 and CAC4A was
1
further studied by H NMR spectroscopy. In the presence of
CAC4A, Rho123 protons showed upfield shifts (Supporting
Information, Figure S4) caused by the ring current effect of
the aromatic nuclei of calixarenes.[19] The shifts of benzene
protons (H4 to H7) are larger than xanthene protons (H1 to
H3), indicating that the benzene part of Rho123 is encapsu-
lated more deeply inside the cavity of CAC4A. The calixarene
protons also underwent upfield shifts affected by Rho123. The
assumed binding geometry was further validated by the 2D
NOESY spectrum (Supporting Information, Figure S5). We
observed the intermolecular correlations between benzene
protons of Rho123 and phenolic protons of CAC4A, and also
correlations between xanthene protons of Rho123 and
substituted benzoic acid protons of CAC4A. Geometry
optimization[20] reveals that the CAC4A–Rho123 complex
assumes a size-matching model. The benzene part of Rho123
locates at the centre of the cavity and xanthene part is at
upper rim (Figure 2a), which is in good agreement with the
NMR results. The optimized structure of CAC4A–Rho123
displays three evident intermolecular hydrogen bonds: two
À
N H···O (1.71 and 1.73 ꢂ) between carboxyl groups of
À
CAC4A and amino groups of Rho123 and one C H···O
Figure 1. a) Molecular structures of CAC4A and Rho123. b) Direct
fluorescence titration of Rho123 (1.0 mm) with CAC4A (up to 8.1 mm)
in PBS buffer (pH 7.4) at 378C and the associated titration curve
(inset) at lem =525 nm and fit according to a 1:1 binding stoichiom-
etry, lex =500 nm.
(2.24 ꢂ) between an aromatic hydrogen of CAC4A and the
carbonyl oxygen of Rho123. To obtain further insights into
the host–guest interactions, the molecular electrostatic poten-
described as having “(almost) unlimited possibilities” due to
their easy derivatization.[16] By directly reacting calix[4]arene
with p-carboxybenzenediazonium chloride at 58C for two
hours, CAC4Awas obtained with quantitative yield (for more
details, see the Supporting Information).[17] Rhodamine 123
(Rho123) is a commercial fluorescent dye and has been
widely used in bioimaging owing to its high brightness and
photostability.[18] Rho123 was employed as the reporter dye
due to the strong complexation with CAC4A and the
corresponding drastic fluorescence quenching (Figure 1b).
A Jobꢀs plot determined the 1:1 binding stoichiometry
between CAC4A and Rho123 (Supporting Information,
Figure S2). The association constant (Ka) was determined as
(1.4 Æ 0.1) ꢁ 107 mÀ1 by fitting the data of the fluorescence
titration (Figure 1b, inset). Such a strong binding is desirable
to reduce unwanted cargo–carrier dissociation of reporter
pair, especially for use in complex biological conditions. In
contrast to covalent methods, the present noncovalent
strategy faces the challenge of the competitive complexation
of biologically coexisting species with CAC4A, which would
lead to release of Rho123 and give rise to imaging noise. The
changes in the fluorescence intensity of CAC4A–Rho123
resulting from biologically important species (adenosine
triphosphate, amino acids, metal ions, glucose, urea, crea-
tinine, and bovine serum albumin) were tested (Supporting
Information, Figure S3). No significant increases in the
Figure 2. a) Optimized binding geometry of the CAC4A–Rho123 com-
plex (right) at the B3LYP-D3/6-31G(d)/SMD(water) level of theory and
MEP-mapped molecular vdW surface of CAC4A (left) and Rho123
(middle). The highest four MEP maxima of Rho123 and the lowest
seven minima of CAC4A are shown by purple and yellow dots,
respectively. Evident hydrogen bonds are shown by dashed lines.
b) dginter =0.007 a.u. isosurfaces colored according to a blue-green-red
scheme over the range À0.05<sign(l2)1<0.05 a.u. for CAC4A–
Rho123 complex (the meaning of dginter and sign(l2)1 can be found in
ref. [22]). Blue indicates a strong attraction, and red indicates a strong
repulsion. c) Frontier orbital energy diagrams and the electron-transfer
path from CAC4A to Rho123.
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tial[21] (MEP) mapped on the van der Waal (vdW) surface of
UV/Vis spectroscopy was employed to examine the
CAC4A and Rho123 was computed (Figure 2a). CAC4A is
electron-rich, especially at the upper rim, and Rho123 is
electron-deficient, especially at the amino groups. The
optimized geometry is favorable because molecules always
tend to interact with each other in a MEP-complementary
manner. The independent gradient model analysis[22] (Fig-
ure 2b) not only validates the existence of three hydrogen
bonds (blue area in isosurfaces) but also reveals p-stacking
interactions (green area in isosurfaces) between CAC4A and
Rho123. Collectively, the high binding affinity between
CAC4A and Rho123 originates from the synergistic effect
among multiple, noncovalent interactions (electrostatic,
hydrogen bonding, and p-stacking).
hypoxia response of CAC4A. CAC4A displays a broad
absorption peak longer than 400 nm (Supporting Informa-
tion, Figure S8), attributed to n-p* transitions of the azo
groups according to the natural transition orbital analysis
(Supporting Information, Table S2). Upon addition of excess
sodium dithionite (SDT), a chemical mimic of azoreduc-
tase,[23,28] a loss of the characteristic yellow color of CAC4A
was detected within 10 min (Photos in Figure 3a). The
The fluorescence quenching of Rho123 upon binding with
CAC4A (Ifree/Ibound) is calculated as 14, which is appropriate
for the subsequent application of hypoxia imaging. The
mechanism of the binding-induced fluorescence quenching is
vital for understanding the supramolecular hypoxia imaging
strategy. Widely studied covalent hypoxia imaging probes rely
on quenching mechanisms of Fçrster resonance energy
transfer (FRET)[11a] or ultrafast conformational change of
the azo part in the excited state.[12] The absorption of CAC4A
shows no appreciable overlap with the emission of Rho123
(Supporting Information, Figure S6), ruling out the FRET
quenching. Moreover, the fluorescence quenching of Rho123
resulting from the conformational change of the azo groups of
CAC4A is unlikely because CAC4A and Rho123 form
a noncovalent complex, not a covalent conjugate.[12,23] Alter-
natively, CAC4A could quench the fluorescence of Rho123
via the photoinduced electron transfer (PET) mechanism.[24]
Calixarenes are well-demonstrated fluorescence quenchers
via the PET mechanism.[25] The PET process between
CAC4A and Rho123 was rationalized by simple molecular
orbital theory,[26] which is a prevalent tool used to discuss the
fluorescence on-off problem.[27] The HOMO energy of
Rho123 is lower than energies of six occupied orbitals
(HOMO to HOMO-5) of CAC4A (Figure 2c and Supporting
Information, Table S1). This results in the occurrence of
reductive-PET, in which an electron on the host transfers to
the guest and fills its singly occupied HOMO. 4-((4-Hydrox-
yphenyl)diazenyl)benzoic acid (CA-phenol; Supporting
Information, Scheme S1), the building subunit of CAC4A,
was employed to explore the superiority of CAC4A.
Although the higher HOMO energy of CA-phenol than
that of Rho123 (Supporting Information, Table S1) permits
the reductive-PET, only very slight quenching of fluorescence
was observed upon adding CA-phenol to Rho123 solution
(Supporting Information, Figure S7). Furthermore, the
absence of quenching of Rho123 by 4-carboxyazobenzene
(Supporting Information, Scheme S1; whose HOMO energy
is lower than that of Rho123, as given in Table S1 in the
Supporting Information) further validates the supposed PET
mechanism (Supporting Information, Figure S7). The above
phenomena indicate the irreplaceable role of calixarene in
reaching two prerequisites of our strategy: 1) the preorgan-
ized skeleton ensures the strong complexation with Rho123;
2) the lower-rim phenolic hydroxyls enable the fluorescence
quenching.
Figure 3. a) Absorbance at 420 nm of CAC4A (10 mm) as a function of
time following addition of SDT (1.0 mm). Inset: Absorbance spectra of
CAC4A (10 mm) before and after reduction by SDT (1.0 mm). b) Rela-
tive fluorescence intensity at 527 nm of CAC4A–Rho123 (30/10 mm) at
different times after addition of SDT (1.0 mm). Inset: Fluorescence
spectra of CAC4A–Rho123 (30/10 mm) before and after reducing by
SDT (1.0 mm). Experimental conditions: PBS buffer, pH 7.4, 378C. The
slight decrease in fluorescence after the sharp increase may be caused
by the partial reduction of Rho123 by the excess SDT. c) Confocal laser
scanning microscopy images of A549 cells incubated with CAC4A–
Rho123 under hypoxic (less than 0.1% O2) or normoxic (20% O2)
conditions for 8 h. The Rho123 emission was obtained using excitation
at 532 nm. Scale bar: 50 mm.
disappearance of the azo absorption indicates a complete
reduction reaction i.e. all four azo groups of CAC4A were
reduced (Figure 3a). The reducing kinetics were quantified by
monitoring the absorbance at 420 nm in real time. The
attenuation curve of the intensity was well fitted in a quasi-
first-order reaction decay model (Adj. R2 > 0.993), giving the
rate constant of 0.905 minÀ1 (Supporting Information, Fig-
ure S9). The half-life was calculated as 46 s, which is at the
same level of similar compounds containing a single azo
group.[29] The reduction product of CAC4A was further
examined by using mass spectrometry analysis. The mass
spectrum of CAC4A shows peaks at 509 and 1017, corre-
sponding to [M+2H]2+ and [M+H]+, respectively (Support-
ing Information, Figure S10a). Aminocalix[4]arene was found
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after incubation with SDT, as shown by the appearance of
peaks at 243, 485, and 969, corresponding to [M+2H]2+,
[M+H]+, and [2M+H]+, respectively (Supporting Informa-
tion, Figure S10b). It indicates that all four azo groups of
CAC4Awere completely cleaved, in accordance with the UV/
Vis results. Besides SDT, CAC4A could also be effectively
and specifically reduced by reduced nicotinamide adenine
dinucleotide phosphate (NADPH) in the presence of DT-
diaphorase, a reductase that is upregulated in many cancers[30]
and activated under hypoxic (Supporting Information, Fig-
ure S11) but not normoxic conditions (Supporting Informa-
tion, Figure S12).
As expected, the fluorescence intensity of Rho123 was
gradually restored in a time-dependent manner and reached
saturation in two minutes with a marked eight-fold increase
after adding SDT to the CAC4A–Rho123 complex (Fig-
ure 3b). Such a pronounced fluorescence increase demon-
strates the feasibility of our host–guest approach in hypoxia
imaging. The working principle is based on the differential
binding between CAC4A and aminocalix[4]arene. CAC4A
shows strong binding to Rho123 (Ka = (1.4 Æ 0.1) ꢁ 107 mÀ1),
while aminocalix[4]arene, as the reduction product, shows
merely weak binding (Ka = (5.4 Æ 0.8) ꢁ 103 mÀ1; Supporting
Information, Figure S13) to Rho123. The Ka decrease of over
three orders of magnitude results in the release of Rho123
and the corresponding fluorescence recovery after reducing
CAC4A. To test the specificity of the hypoxia response, the
fluorescence recovery was investigated by replacing SDTwith
other cellular reductants (cysteine, glutathione, reduced
nicotinamide adenine dinucleotide, NADPH, and homocys-
teine) under both hypoxic and normoxic conditions (Support-
ing Information, Figure S14). No fluorescence enhancement
was observed, indicating that the CAC4A–Rho123 complex is
specifically responsive to hypoxia. Moreover, the acid toler-
ance of the reporter pair was investigated. At pH 5.0, CAC4A
under normoxic and hypoxic conditions (Supporting Infor-
mation, Figure S16) for 24 h. Generally, even when loaded
with a relatively high concentration of 50 mm, A549 cells
exhibited a survival rate greater than 90% after incubation
for 24 h under both normoxic and hypoxic conditions. These
results suggest that under our experimental conditions (30 mm
for 8 h incubation), CAC4A exhibited negligible cytotoxic
effect against cells.
In conclusion, we demonstrate a new noncovalent strategy
for imaging hypoxia based on the concept of supramolecular
chemistry, which was validated in living cells. Compared with
widely used covalent strategies, the present noncovalent
strategy based on host–guest chemistry exhibits several
intrinsic advantages: 1) the use of commercial probes without
time- and cost-consuming synthesis; 2) releasing probes with
high fidelity under hypoxia; 3) easy adaptability to other
probes (and even treating agents) for constructing a universal
platform. Such a noncovalent strategy is easily amenable to
other azo-containing macrocycles for not only hypoxia-
responsive imaging but also drug delivery. Noticeably, over
1200 azo-containing macrocycles have been reported up to
now;[32] however, no hypoxia-responsive application has ever
been demonstrated. This work not only paves a new way to
imaging hypoxia but also explores new biomedical applica-
tions of azo macrocycles.
Acknowledgements
This work was supported by NSFC (51873090 and 21672112),
the Fundamental Research Funds for the Central Universities
and the Program of Tianjin Young Talents, which are grate-
fully acknowledged. The authors also thank Debin Zheng at
College of Life Sciences, Nankai University for the mass
spectrometry experiments.
also binds strongly to Rho123 (Ka = (1.5 Æ 0.4) ꢁ 107 mÀ1
;
Supporting Information, Figure S15). The comparable affin-
ities between pH 7.4 and 5.0 eliminate the possibility of the
undesired release of Rho123 triggered by acidic environ-
ments, which is meaningful for the following cell experiments
when taking acidic conditions in the endosome/lysosome
(about pH 5.0) into account.
Conflict of interest
The authors declare no conflict of interest.
Keywords: azo compound · calixarene · host–guest complex ·
With the aforementioned results in hand, the application
of the CAC4A–Rho123 reporter pair in imaging hypoxia was
explored. As a proof-of-concept, the utilization of CAC4A–
Rho123 in hypoxia-selective imaging in living cells was
investigated. A549 cancer cells were incubated with
CAC4A–Rho123 in both normoxic and hypoxic conditions.
As shown in Figure 3c, incubation of A549 cells with
CAC4A–Rho123 under normoxia shows very low fluores-
cence signal. In contrast, when the cells were cultured under
hypoxia, much brighter fluorescence was observed within the
cells. These results prove that the CAC4A–Rho123 reporter
pair was able to detect hypoxia in living cells.
Cellular probes for practical applications should mini-
mally perturb living systems at the concentrations used. The
cytotoxicity of CAC4A was evaluated by 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays[31]
with various concentrations of CAC4A from 10 to 50 mm,
hypoxia imaging · supramolecular chemistry
[1] a) J. M. Brown, W. R. Wilson, Nat. Rev. Cancer 2004, 4, 437 –
447; b) R. A. Gatenby, R. J. Gillies, Nat. Rev. Cancer 2008, 8, 56 –
61.
[2] Q. Chang, I. Jurisica, T. Do, D. W. Hedley, Cancer Res. 2011, 71,
3110 – 3120.
[3] B. A. Teicher, Cancer Metastasis Rev. 1994, 13, 139 – 168.
[4] Z. Huang, H. Xu, A. D. Meyers, A. I. Musani, L. Wang, R. Tagg,
A. B. Barqawi, Y. K. Chen, Technol. Cancer Res. Treat. 2008, 7,
309 – 320.
[5] a) L. Schito, G. L. Semenza, Trends Cancer 2016, 2, 758 – 770;
b) P. Vaupel, Oncologist 2008, 13, 21 – 26.
[6] a) A. M. Jubb, F. M. Buffa, A. L. Harris, J. Cell. Mol. Med. 2010,
14, 18 – 29; b) H. Minn, T. Gronroos, G. Komar, O. Eskola, K.
Lehtio, J. Tuomela, M. Seppanen, O. Solin, Curr. Pharm. Des.
4
www.angewandte.org
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
These are not the final page numbers!

Angewandte
Communications
Chemie
2008, 14, 2932 – 2942; c) J. G. Rajendran, K. R. Hendrickson,
[16] a) V. Bçhmer, Angew. Chem. Int. Ed. Engl. 1995, 34, 713 – 745;
A. M. Spence, M. Muzi, K. A. Krohn, D. A. Mankoff, Eur. J.
Nucl. Med. Mol. Imaging 2006, 33, 44 – 53; d) H. Shi, X. Ma, Q.
Zhao, B. Liu, Q. Qu, Z. An, Y. Zhao, W. Huang, Adv. Funct.
Mater. 2014, 24, 4823 – 4830; e) Y. Zeng, Y. Liu, J. Shang, J. Ma,
R. Wang, L. Deng, Y. Guo, F. Zhong, M. Bai, S. Zhang, D. Wu,
Plos One 2015, 10, e0121293.
Angew. Chem. 1995, 107, 785 – 818; b) M. A. Beatty, J. Borges-
Gonzalez, N. J. Sinclair, A. T. Pye, F. Hof, J. Am. Chem. Soc.
2018, 140, 3500 – 3504.
[17] Y. Morita, T. Agawa, E. Nomura, H. Taniguchi, J. Org. Chem.
1992, 57, 3658 – 3662.
[18] V. Reshetnikov, S. Daum, C. Janko, W. Karawacka, R. Tietze, C.
Alexiou, S. Paryzhak, T. Dumych, R. Bilyy, P. Tripal, B. Schmid,
R. Palmisano, A. Mokhir, Angew. Chem. Int. Ed. 2018, 57,
11943 – 11946; Angew. Chem. 2018, 130, 12119 – 12122.
[19] a) D.-S. Guo, H.-Q. Zhang, F. Ding, Y. Liu, Org. Biomol. Chem.
2012, 10, 1527 – 1536; b) Z. Zheng, W.-C. Geng, J. Gao, Y.-Y.
Wang, H. Sun, D.-S. Guo, Chem. Sci. 2018, 9, 2087 – 2091.
[20] a) P. J. Stephens, F. J. Devlin, C. F. Chabalowski, M. J. Frisch, J.
Phys. Chem. 1994, 98, 11623 – 11627; b) A. D. Becke, J. Chem.
Phys. 1993, 98, 5648 – 5652; c) P. C. Hariharan, J. A. Pople,
Theor. Chim. Acta 1973, 28, 213 – 222; d) W. J. Hehre, R.
Ditchfield, J. A. Pople, J. Chem. Phys. 1972, 56, 2257 – 2261.
[21] J. S. Murray, P. Politzer, WIREs Comput. Mol. Sci. 2011, 1, 153 –
163.
[22] C. Lefebvre, G. Rubez, H. Khartabil, J. C. Boisson, J. Contreras-
Garcia, E. Henon, Phys. Chem. Chem. Phys. 2017, 19, 17928 –
17936.
[23] A. Chevalier, C. Mercier, L. Saurel, S. Orenga, P. Y. Renard, A.
Romieu, Chem. Commun. 2013, 49, 8815 – 8817.
[24] J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
Springer US, New York, 2006.
[25] a) R. N. Dsouza, U. Pischel, W. M. Nau, Chem. Rev. 2011, 111,
7941 – 7980; b) D.-S. Guo, V. D. Uzunova, X. Su, Y. Liu, W. M.
Nau, Chem. Sci. 2011, 2, 1722 – 1734.
[26] A. Weller, Pure Appl. Chem. 1968, 16, 115 – 123.
[27] A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M.
Huxley, C. P. McCoy, J. T. Rademacher, T. E. Rice, Chem. Rev.
1997, 97, 1515 – 1566.
[28] a) G. Leriche, G. Budin, L. Brino, A. Wagner, Eur. J. Org. Chem.
2010, 4360 – 4364; b) Y. Y. Yang, M. Grammel, A. S. Raghavan,
G. Charron, H. C. Hang, Chem. Biol. 2010, 17, 1212 – 1222.
[29] P. Verwilst, J. Han, J. Lee, S. Mun, H.-G. Kang, J. S. Kim,
Biomaterials 2017, 115, 104 – 114.
[30] a) J. Rao, A. Khan, J. Am. Chem. Soc. 2013, 135, 14056 – 14059;
b) J. Rao, C. Hottinger, A. Khan, J. Am. Chem. Soc. 2014, 136,
5872 – 5875; c) R. Li, M. A. Bianchet, P. Talalay, L. M. Amzel,
Proc. Natl. Acad. Sci. USA 1995, 92, 8846 – 8850.
[31] T. Mosmann, J. Immunol. Methods 1983, 65, 55 – 63.
[32] W.-C. Geng, H. Sun, D.-S. Guo, J. Inclusion Phenom. Macro-
cyclic Chem. 2018, 92, 1 – 79.
[7] S. Kizaka-Kondoh, M. Inoue, H. Harada, M. Hiraoka, Cancer
Sci. 2003, 94, 1021 – 1028.
[8] a) K. Mikami, M. Naito, A. Tomida, M. Yamada, T. Sirakusa, T.
Tsuruo, Cancer Res. 1996, 56, 2823 – 2826; b) Y. Shen, Q. Zhang,
X. Qian, Y. Yang, Anal. Chem. 2015, 87, 1274 – 1280.
[9] a) A. Sharma, J. F. Arambula, S. Koo, R. Kumar, H. Singh, J. L.
Sessler, J. S. Kim, Chem. Soc. Rev. 2019, https://doi.org/10.1039/
C8CS00304A; b) X. Li, N. Kwon, T. Guo, Z. Liu, J. Yoon,
Angew. Chem. Int. Ed. 2018, 57, 11522 – 11531; Angew. Chem.
2018, 130, 11694 – 11704.
[10] a) J. Zhou, W. Shi, L.-H. Li, Q.-Y. Gong, X.-F. Wu, X.-H. Li, H.-
M. Ma, Chem. Asian J. 2016, 11, 2719 – 2724; b) J. Zhang, H.-W.
Liu, X.-X. Hu, J. Li, L.-H. Liang, X.-B. Zhang, W. Tan, Anal.
Chem. 2015, 87, 11832 – 11839; c) Q.-Q. Wan, X.-H. Gao, X.-Y.
He, S.-M. Chen, Y.-C. Song, Q.-Y. Gong, X.-H. Li, H.-M. Ma,
Chem. Asian J. 2014, 9, 2058 – 2062; d) T. Guo, L. Cui, J. Shen, W.
Zhu, Y. Xu, X. Qian, Chem. Commun. 2013, 49, 10820 – 10822;
e) L. Cui, Y. Zhong, W. Zhu, Y. Xu, Q. Du, X. Wang, X. Qian, Y.
Xiao, Org. Lett. 2011, 13, 928 – 931; f) H. S. Kim, A. Sharma,
W. X. Ren, J. Han, J. S. Kim, Biomaterials 2018, 185, 63 – 72.
[11] a) K. Kiyose, K. Hanaoka, D. Oushiki, T. Nakamura, M.
Kajimura, M. Suematsu, H. Nishimatsu, T. Yamane, T. Terai,
Y. Hirata, T. Nagano, J. Am. Chem. Soc. 2010, 132, 15846 –
15848; b) A. Chevalier, W. Piao, K. Hanaoka, T. Nagano, P.-Y.
Renard, A. Romieu, Methods Appl. Fluoresc. 2015, 3, 044004;
c) Q. Cai, T. Yu, W. Zhu, Y. Xu, X. Qian, Chem. Commun. 2015,
51, 14739 – 14741; d) L. Sun, G. Li, X. Chen, Y. Chen, C. Jin, L. Ji,
H. Chao, Sci. Rep. 2015, 5, 14837; e) F. Perche, S. Biswas, T.
Wang, L. Zhu, V. P. Torchilin, Angew. Chem. Int. Ed. 2014, 53,
3362 – 3366; Angew. Chem. 2014, 126, 3430 – 3434; f) A. Popat,
B. P. Ross, J. Liu, S. Jambhrunkar, F. Kleitz, S. Z. Qiao, Angew.
Chem. Int. Ed. 2012, 51, 12486 – 12489; Angew. Chem. 2012, 124,
12654 – 12657; g) P. Yuan, H. Zhang, L. Qian, X. Mao, S. Du, C.
Yu, B. Peng, S. Q. Yao, Angew. Chem. Int. Ed. 2017, 56, 12481 –
12485; Angew. Chem. 2017, 129, 12655 – 12659.
[12] W. Piao, S. Tsuda, Y. Tanaka, S. Maeda, F. Liu, S. Takahashi, Y.
Kushida, T. Komatsu, T. Ueno, T. Terai, T. Nakazawa, M.
Uchiyama, K. Morokuma, T. Nagano, K. Hanaoka, Angew.
Chem. Int. Ed. 2013, 52, 13028 – 13032; Angew. Chem. 2013, 125,
13266 – 13270.
[13] M. I. Uddin, S. M. Evans, J. R. Craft, L. J. Marnett, M. J. Uddin,
A. Jayagopal, ACS Med. Chem. Lett. 2015, 6, 445 – 449.
[14] a) X. Ma, Y. Zhao, Chem. Rev. 2015, 115, 7794 – 7839; b) J. Gao,
J. Li, W.-C. Geng, F.-Y. Chen, X. Duan, Z. Zheng, D. Ding, D.-S.
Guo, J. Am. Chem. Soc. 2018, 140, 4945 – 4953.
Manuscript received: November 23, 2018
Revised manuscript received: January 8, 2019
Accepted manuscript online: January 10, 2019
Version of record online: && &&, &&&&
[15] A. Norouzy, Z. Azizi, W. M. Nau, Angew. Chem. Int. Ed. 2015,
54, 792 – 795; Angew. Chem. 2015, 127, 804 – 808.
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Imaging Agents
A supramolecular strategy for fluorescent
hypoxia imaging is proposed based on
the host–guest complexation of azoma-
crocycles with commercial dyes.
W.-C. Geng, S. Jia, Z. Zheng, Z. Li,
D. Ding, D.-S. Guo*
&&& — &&&
A Noncovalent Fluorescence Turn-on
Strategy for Hypoxia Imaging
6
www.angewandte.org
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
These are not the final page numbers!