Molecular Chemiluminescent Probes with a Very Long Near-Infrared Emission Wavelength for in Vivo Imaging

Article abstract of DOI:10.1002/anie.202013531

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, β-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues.

Full text of DOI:10.1002/anie.202013531

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Accepted Article
Title: Molecular Chemiluminescent Probes with a Record Long Near-
infrared Turn-on Wavelength for In vivo Imaging
Authors: Kanyi Pu, Jingsheng Huang, Yuyan Jiang, Jingchao Li, and
Jiaguo Huang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013531
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10.1002/anie.202013531
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Molecular Chemiluminescent Probes with a Record Long Near-
infrared Turn-on Wavelength for In vivo Imaging
Jingsheng Huang,† Yuyan Jiang,† Jingchao Li, Jiaguo Huang and Kanyi Pu*
[*]
J. Huang, Y. Jiang, Dr. J. Li, J. Huang, Prof. K. Pu
School of Chemical and Biomedical Engineering
Nanyang Technological University
70 Nanyang Drive, Singapore 637457 (Singapore)
E-mail: kypu@ntu.edu.sg
These authors contributed equally.
[†]
Supporting information for this article is given via a link at the end of the document.
Abstract: Chemiluminescence imaging is imperative for diagnostics
and imaging due to its intrinsically high sensitivity. To improve in vivo
detection of biomarkers, chemiluminophores that simultaneously
possess near-infrared (NIR) emission and modular structures
amenable to construction of activatable probes are highly desired,
which however are rare. We herein report two chemiluminophores
with record long NIR emission (> 750 nm) via integration of
dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-
constructed by caging the phenol group with biomarker-cleavable
moieties.[14] Such
generic molecular design enables
ultrasensitive chemiluminescence imaging of various enzymes
(e.g. γ-glutamyl
nitroreductase,[15] transpeptidase,[16]
cathepsins,[17] etc.[18-19]) and reactive oxygen species (ROS) (e.g.
hydrogen peroxide,[20] superoxide anion,[21] peroxynitrite,[22] etc.)
and formaldehyde.[23] However, the majority of these probes emit
in visible region with limited tissue penetration depth.[24]
a
benzoselenopyran
with
dioxetane
unit.
Caging
of the
To shift the chemiluminescence towards
biological transparent near-infrared (NIR)
a
relatively
region,
chemiluminophores with different cleavable moieties leads to NIR
chemiluminescence probes (NCPS) that only activate signals upon
reaction with reactive oxygen species or enzymes with the tissue-
penetration depth up to 2 cm. Particularly, NCPSg can trigger its
chemiluminescence at 760 nm for sensitive detection of β-
galactosidase (a cancer biomarker), allowing to image and
differentiate the expression levels of β-galactosidase in different
cancer cells in living mouse. Thus, this study provides NIR
chemiluminescence molecular scaffolds applicable for in vivo turn-on
imaging of versatile biomarkers in deep tissues.
chemiluminescent substrates have been doped into nanoparticles
to construct resonance energy transfer.[25-29] However, such
nanoparticle approach can potentially reduce chemiluminescent
brightness as a result of extra energy relay processes, and the
physical entrapment of chemiluminescent substrate significantly
prevents its contact with biomolecules, limiting their detection
species to small and diffusive molecules such as ROS. Thus,
direct modification
of Shaap’s dioxetane into NIR
chemiluminescence scaffolds is a desired strategy to further
improve and broaden the application of chemiluminescence
imaging.[30-31] However, there are only four dioxetane-based
chemiluminophores with the NIR emission ranging from 660 to
714 nm (Table S1).
Optical imaging has become an indispensable tool in fundamental
research and clinical practice.[1] However, conventional
fluorescence
imaging
suffers
from
inevitable
tissue
autofluorescence caused by light excitation, which severely
compromises imaging depth and sensitivity.[2] To address this
issue, excitation-free luminescent imaging modalities have been
increasingly explored with the focus on bioluminescence,[3]
chemiluminescence,[4] afterglow and persistent luminescence
imaging.[5-6] These modalities obviate the need for real-time light
excitation by using chemical reactions or physical lattice defects
to produce luminescent output,[7] affording minimized background
noise and enhanced tissue-penetration depth.[8] Thus, these
excitation-free imaging modalities have been extensively utilized
in
a broad spectrum of biomedical applications including
ultrasensitive bioassays,[9] disease diagnosis,[10] and imaging-
guided therapies.[11]
Distinct
from
other
excitation-free
modalities,
Figure 1. (a) Chemical structure of chemiluminophores (DCBP-O, DCBP-S and
DCBP-Se). (b) HOMO-LUMO levels of DCBP-O, DCBP-S and DCBP-Se. (c)
Synthetic routes for NCPX (X=O, S or Se). Reagents and conditions: (i) DBP,
DBTP or DBSeP (Scheme S1), piperidine, CH3CN, N2, reflux, 2 h; (ii) 10-X (X =
chemiluminescence imaging is of particular interest due to its
feasibility to develop activatable probes without the reliance on
extra factors (e.g. luciferase for bioluminescence, or in situ photo-
charging for persistent luminescence).[12-13] On the basis of
O,
S or Se), HCOOH, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dry
CH2Cl2, r.t., 4-dimethylaminopyridine, overnight; (xi) 11 (X = O, S or Se),
methylene blue, THF/MeOH (2/1, v/v), 0 oC, 6 h.
Schaap’s
adamantylidene-1,2-dioxetane,
a
series
of
chemiluminescent activatable probes that only trigger their
chemiluminescence in the presence of biomarkers have been
1
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10.1002/anie.202013531
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We herein report the synthesis of two new
chemiluminophores with record long NIR emission wavelengths
and their development into molecular chemiluminescence probes
for in vivo imaging. As shown in Figure 1a, the
chemiluminophores named DBP-phenoxy-dioxetane (DPD-O),
DBTP-phenoxy-dioxetane (DPD-S) and DBSeP-phenoxy-
dioxetane (DPD-Se) are composed of NIR fluorophores based on
dicyanometh-ylene-4H-benzopyran (DBP), dicyanomethylene-
were much smaller, ~ 7.1, 7.5 and 7.8-fold for NCPO, NCPS and
NCPSe, respectively (Figure S2b). This was mainly attributed to
the higher background of fluorescence relative to the
chemiluminescence of these probes. The bathochromic emission
maxima of NCPS and NCPSe relative to NCPO coincided well with
the result of DFT calculation that NCPS and NCPSe had smaller
HOMO-LUMO energy gaps than NCPO (Figure 1a&b). This
activation mechanism was further validated by high-performance
liquid chromatography (HPLC), indicating the transformation of
NCPX to the corresponding activated form ANCPX via ONOO−
mediated nucleophilic addition followed by hydrolysis (Figure S3).
4H-benzothiopyran
(DBTP),
and
dicyanomethylene-4H-
benzoselenopyran (DBSeP), respectively. Note that DPD-O was
first reported by Shabat’s group, showing the highest
chemiluminescence
compared
to
other
NIR
chemiluminophores.[30] The density functional theory (DFT)
calculation (Figure 1b) revealed that substitution of oxygen atom
with sulfur and selenium reduced the energy gaps from 2.61 to
2.42 and 2.41 eV, respectively. The narrowed bandgaps should
be ascribed to the increased atomic radius and change in
electronegativity,[32] implying the potential of red-shifted NIR
chemiluminescence via this atomic alternation approach.
Encouraged by the DFT results, three activatable NIR
chemiluminescent probes (NCPO, NCPS and NCPSe) were
synthesized (Figure 1c), whose phenol groups were caged with
peroxynitrite (ONOO−) cleavable moiety. DBP, DBTP, DBSeP
and intermediate compound 6 were synthesized according to
Scheme S1. 2-Methylchromone (1) was synthesized by
cyclization reaction between 2-hydroxyacetophenone and ethyl
acetate in the presence of concentrated HCl. The reaction of
benzenethiol with ethyl benzoylacetate in the polyphosphoric acid
gave 2-methyl-thiochroman-4-one (2). Differently, 2-methyl-
selenochroman-4-one (5) was synthesized in multiple steps.
Firstly, 2-bromobenzaldehyde was reacted via 1,2-addition
reaction in 1-propynylmagnesium bromide solution to afford
compound 3. Oxidation of 3 by Iodoxybenzoic acid gave the
compound 4, which was reacted with NaBH4 and Se powder to
yield (5). DBP, DBTP and DBSeP were all synthesized via
condensation of the corresponding chromone with malononitrile.
Then, Knoevenagel condensation reaction between compounds
DBP, DBTP or DBSeP, and compound 6 led to compounds 7-X
(X = O, S or Se), which were subsequently caged with ONOO−-
responsive formate to yield the precursor 8-X. At last, NCPX
(NCPO, NCPS or NCPSe) was synthesized by oxidation of
compound 8-X with singlet oxygen (1O2) using methylene blue as
a photosensitizer.
The optical properties and sensing abilities of NCPX were
measured and compared. In the absence of ONOO−, all the
probes had similar maxima absorption at ~450 nm (Figure S1).
Moreover, they had low fluorescence, and negligible
chemiluminescence (Figure S2 and 1), because these
unactivated probes were in a “caged” state wherein the electron-
donating ability of the oxygen atom of phenol group was
diminished. Upon addition of ONOO−, the absorption maxima of
NCPO, NCPS and NCPSe red-shifted to ~ 525, 545 and 585 nm
(Figure S1), respectively; meanwhile, their chemiluminescence
appeared with the emission maxima at 660, 760 and 780 nm,
respectively (Figure 2a), with the intensities that were ~ 1261,
1209 and 1240-fold stronger than their corresponding inactive
forms. NCPX is cleavage to form DPD-X intermediate, then
spontaneously decompose to emit NIR light via CIEEL
mechanism.[31] Although the chemiluminescence profiles of these
probes corresponded well with their fluorescence spectra (Figure
S2a), the fluorescence enhancements after ONOO− activation
Figure 2. (a) Chemiluminescence spectra of NCPX (x=O, S or Se), (5 μM) in the
presence of ONOO– (10 μM) in PBS (10 mM, pH=7.4, 1% DMSO) at 37 oC. (b)
Chemiluminescence enhancements of NCPX (5 μM) after addition of different
ROS (10 μM) in PBS (10 mM, pH 7.4) or metal ions (100 μM) at 37 oC. (c)
Chemiluminescence images of NCPX (10 µM) in PBS after the addition ONOO–
(20 µM) through chicken breast tissues with different thicknesses. (d) SBRs for
NCPX as a function of tissue depth. Data are the mean ± SD; n=3 independent
experiments.
The selectivity of the probes towards ONOO− was studied. All
NCPX exhibited negligible optical responses in the presence of
other ROS (H2O2, ClO−, •OH, 1O2 and O2•−) or metal ions (K+, Ca2+,
Fe2+ and Mg2+). On the basis of chemiluminescent readouts,
NCPX had similar limits of detection (LODs) toward ONOO− at
~14-18 nM (Figure S2c), lower than the previously reported
fluorescence probes (~ above 100 nM) and in the similar range
for existing chemiluminescence probes (6 to 17 nM).[34-36], [22] In
addition, the chemiluminescence half-lives of NCPX (74 s for
NCPO, 62 s for NCPS and 56 s for NCPSe) were long enough for
real-time imaging in living subjects (Figure S2d).
To evaluate the potential of NCPX for in vivo imaging, tissue
penetration depths of these probes were measured and
compared. In the presence of excessive ONOO−, the
chemiluminescence of NCPX was recorded at 760 nm through
chicken breast tissues with different thicknesses (Figure 2c).
Without tissues, NCPS intrinsically had the brightest
chemiluminescence and the highest signal to background ratio
(SBR) (~415.1) among three probes (Figure 2d). This was
attributed to the fact that among these probes, NCPS had the
highest chemiluminescence intensity at 760 nm despite its
2
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10.1002/anie.202013531
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chemiluminescence quantum yield was lower than NCPO (Table
S2 and Figure S4). With the increase of tissue thickness, the
chemiluminescence signals from all NCPX were gradually
attenuated. The chemiluminescence of NCPS was detectable up
to 2 cm, whereas that of others was hardly detectable at 1.5 cm
(Figure 2c). At the tissue depth of 1.5 cm, the SBR for NCPS (68.6)
was 3.7- and 1.9-fold higher than that for NCPO (18.3) and NCPSe
(36.9), respectively. The superior SBRs in deeper tissues of NCPO
should be attributed to its red-shifted NIR emission maximum and
higher chemiluminescence at 760 nm relative to others. These
data indicated that NCPS outperformed NCPO and NCPSe in terms
of both tissue penetration depth and sensitivity, implying its
superior performance for in vivo imaging.
mU/L (Figure S5), lower than the majority of reported
fluorescence and chemiluminescence probes (170 to 4000
mU/L).[31], [38-39]
The cytocompatibility of NCPSg was validated for both β-gal-
positive ovarian cancer cells (SKOV3) and β-gal-negative control
cells (HeLa).[40] NCPSg induced no obvious cytotoxicity to both
cells (Figure S6). After incubation of the cells with NCPSg for 30
min, the chemiluminescence was detected for SKOV3 cells,
which was 13-fold higher than that of HeLa cells (Figure 4b).
Similarly, the NIR fluorescence intensity of SKOV3 cells was 3.5-
fold higher than that of Hela cells (Figure 4a and 4b). When
SKOV3 cells were pretreated with β-gal inhibitor (D-galactose),
both chemiluminescence and fluorescence signals decreased to
the basal levels. (Figure 4b). These results were consistent with
the fact that SKOV3 cells overexpress high level of β-gal while
HeLa cells barely express β-gal,[40] and validated the feasibility of
NCPSg to sensitively detect β-gal in living cells.
The capability of NCPSg for in vivo imaging of β-gal was
examined on SKOV3 or HeLa tumor-bearing nude mice. After
intratumoral injection of NCPSg (32 μM kg-1), both
chemiluminescence and fluorescence were longitudinally
recorded (Figure 4c). Chemiluminescence signal from tumor
region in SKOV3 tumor-bearing mice gradually increased and
reached the maximum at 20 min post-injection (Figure 4d). At this
timepoint, the chemiluminescence intensity of SKOV3 tumor was
6.5-fold stronger than that from HeLa tumor. Furthermore, the
chemiluminescence of SKOV3 tumor was close to the basal level
if the mice were pre-treated with the β-gal inhibitor. In comparison,
the fluorescence of NCPSg failed to detect β-gal in tumor (Figure
S7), due to the strong tissue autofluorescence caused by short-
wavelength excitation at 450 nm.[24] After imaging, major organs
from NCPSg-treated living mice were harvested for histological
studies. Hematoxylin and eosin (H&E) staining indicated no
noticeable histopathological damage in major organs of mice
(Figure S8), confirming the good biocompatibility of NCPSg.
Figure 3. (a) Scheme for chemiluminescence detection of β-gal using NCPSg
.
(b) Chemiluminescence spectra of NCPSg before and after incubation with β-gal
(1 U/mL) in 10 mM PBS (pH = 7.4, 2% DMSO 10 mM MgCl2). (c) Fluorescence
and chemiluminescence enhancements of NCPSg (5 μM) in the presence of β-
gal (1 U/mL), other different enzymes (1 U/mL), or β-gal (1 U/mL) with inhibitor
(100 mM) in PBS (10 mM, pH 7.4) at 37 oC. Inset: the corresponding optical
images of NCPSg with different enzymes. (d) HPLC analysis of NCPSg (5 μM)
after incubation with β-gal (1 U/ml).
The optimal chemiluminophores (DPD-S) was modified into
the activatable probe (NCPSg) for detection of the enzymatic
activity of β-galactosidase (β-gal). β-gal is a glycoside hydrolase
and overexpressed in a panel of ovarian cancers.[37] The
synthesis of β-gal-responsive probe NCPSg is shown in Scheme
S1. Compound 7-S was firstly caged by tetra-O-acetyl-α-D-
galactopyranosyl-1-bromide, and then deprotected to give
1
compound 9-S, which was oxidized by O2 to yield NCPSg. After
incubation with β-gal for 30 min, NCPSg displayed ~ 85.6-fold
chemiluminescence and 5.5-fold fluorescence enhancements at
760 nm (Figure 3b, 3c); by contrast, the signal enhancement of
NCPSg was substantially compromised in the presence of a
galactosidase inhibitor (D-galactose) (Figure 3c). Such a β-gal-
induced chemiluminescent activation was further verified with
HPLC, showing
a newly emerged peak assigned to the
corresponding activated compound ANCPS at 10.8 min (Figure
3d). Moreover, NCPSg showed superior selectivity to β-gal over
other enzymes such as cathepsin B (Cat B), gamma-glutamyl
transferase (GGT), caspase 3 (Casp-3), alanine aminopeptidase
(AAP), furin, dipeptidyl peptidase-4 (DPPIV) (Figure 3d). In
addition, the LOD of NCPSg towards β-gal was measured to be 73
Figure 4. (a) NIR chemiluminescence and fluorescence imaging of Hela and
SKOV3 cells after incubation with NCPSg (5 μM) for 30 min. The inhibitor group:
the SKOV3 cells treated with inhibitor (D-galactose, 100 mM kg-1) for 1 h before
incubation with NCPSg as the inhibitor group. (b) Mean NIR chemiluminescence
and fluorescence intensities in (a). (c) Representative NIR chemiluminescent
images of living mice acquired at 10, 20 or 50 min after intratumor injection of
NCPSg (32 μM kg-1). (d) The dynamic chemiluminescent intensities of NCPSg in
3
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the tumor of living mice at different post-injection timepoints. n.s.: not significant;
**p < 0.01 (n=3).
J. D. Stevenson, A. Dietel, N. R. Thomas, ChemComm 1999, 2105-2106;
d) R. C. Buxton, B. Edwards, R. R. Juo, J. C. Voyta, M. Tisdale, R. C.
Bethell, Anal. Biochem. 2000, 280, 291-300.
[15] L. S. Ryan, J. Gerberich, J. Cao, W. An, B. A. Jenkins, R. P. Mason, A.
R. Lippert, ACS Sens. 2019, 4, 1391-1398.
In conclusion, we have reported two new chemiluminophores
(DPD-S and DPD-Se) with the respective emission maxima at 760
and 780 nm, longer than existing chemiluminescent substrates. In
conjunction with the high brightness at 760 nm, the
chemiluminescence of DPD-S based probe enabled deeper
tissue penetration relative to the other two analogs. By caging its
phenol group using different responsive moieties, DPD-S was
developed into molecular chemiluminescence probes (NCPS and
NCPSg) for respective detection of ONOO− and β-gal with high
sensitivity and selectivity. Moreover, NCPSg was validated in cells
and tumor mouse model, proving its ability to sensitively detect β-
gal and differentiate its expression levels in different cells. Thus,
our study provides an atomic alternation approach to develop NIR
chemiluminophores that can serve as the general molecular
scaffolds for in vivo turn-on optical imaging of biomarkers.
[16] R. An, S. Wei, Z. Huang, F. Liu, D. Ye, Anal. Chem. 2019, 91, 13639-
13646.
[17] M. Roth-Konforti, C. Bauer, D. Shabat, Angew. Chem. Int. Ed. 2017, 56,
15633-15638; Angew. Chem. 2017, 129, 15839-15844.
[18] S. Son, M. Won, O. Green, N. Hananya, A. Sharma, Y. Jeon, J. H. Kwak,
J. L. Sessler, D. Shabat, J. S. Kim, Angew. Chem. Int. Ed. 2019, 58,
1739-1743; Angew. Chem. 2019, 131, 1753-1757.
[19] Z. Hai, J. Li, J. Wu, J. Xu, G. Liang, J. Am. Chem. Soc. 2017, 139, 1041-
1044.
[20] S. Ye, N. Hananya, O. Green, H. Chen, A. Q. Zhao, J. Shen, D. Shabat,
D. Yang, Angew. Chem. Int. Ed. 2020, 59, 14326-14330; Angew. Chem.
2020, 132, 14432-14436.
[21] a) J. Huang, J. Li, Y. Lyu, Q. Miao, K. Pu, Nat. Mater. 2019, 18, 1133-
1143; b) P. Cheng, Q. Miao, J. Li, J. Huang, C. Xie, K. Pu, J. Am. Chem.
Soc. 2019, 141, 10581-10584.
[22] J. Huang, J. Huang, P. Cheng, Y. Jiang, K. Pu, Adv. Funct. Mater. 2020,
30, 2003628.
Acknowledgements
[23] K. J. Bruemmer, O. Green, T. A. Su, D. Shabat, C. J. Chang, Angew.
Chem. Int. Ed. 2018, 57, 7508-7512; Angew. Chem. 2018, 130, 7630-
7634.
This work was supported by Nanyang Technological University
(Start-up Grant: M4081627) and Singapore Ministry of Education
Academic Research Fund Tier 1 (2019-T1-002-045, RG125/19)
and Tier 2 (MOE2018-T2-2-042).
[24] J. Huang, K. Pu, Angew. Chem. Int. Ed. 2020, 59, 11717-11731; Angew.
Chem. 2020, 132,11813-11827.
[25] Y. Yang, S. Wang, L. Lu, Q. Zhang, P. Yu, Y. Fan, F. Zhang, Angew.
Chem. Int. Ed. 2020, 59, 18380-18385; Angew. Chem. 2020, 132,18538-
18543.
Keywords: chemiluminescence • molecular probes • in vivo
[26] X. Ni, X. Zhang, X. Duan, H. L. Zheng, X. S. Xue, D. Ding, Nano Lett.
2018, 19, 318-330.
bioimaging •
[27] X. Zhen, C. Zhang, C. Xie, Q. Miao, K. L. Lim, K. Pu, ACS Nano 2016,
10, 6400-6409.
[1]
a) B. R. Smith, S. S. Gambhir, Chem. Rev. 2017, 117, 901-986; b)
Mariscal, A. Garcia, M. Carnero, E. Gomez, J. Fernandez-Crehuet,
BioTechniques. 1994, 16, 888-893.
[28] A. J. Shuhendler, K. Pu, L. Cui, J. P. Uetrecht, J. Rao, Nat. Biotechnol.
2014, 32, 373.
[29] D. Cui, J. Li, X. Zhao, K. Pu, R. Zhang, Adv. Mater. 2020, 32, 1906314.
[30] a) O. Green, S. Gnaim, R. Blau, A. Eldar-Boock, R. Satchi-Fainaro, D.
Shabat, J. Am. Chem. Soc. 2017, 139, 13243-13248; b) D. Shabat, O.
Green, WO 2018216013A1, 2018.
[2]
[3]
a) J. Li, K. Pu, Chem. Soc. Rev. 2019, 48, 38-71; b)Y. Jiang, K. Pu, Acc.
Chem. Res. 2018, 51, 1840-1849;.
Y. Su, J. R. Walker, Y. Park, T. P. Smith, L. X. Liu, M. P. Hall, L. Labanieh,
R. Hurst, D. C. Wang, L. P. Encell, N. Kim, F. Zhang, M. A. Kay, K. M.
Casey, R. G. Majzner, J. R. Cochran, C. L. Mackall, T. A. Kirk-land, M.
Z. Lin, Nat. Methods 2020, 17, 852-860.
[31] N. Hananya, A. Eldar Boock, C. R. Bauer, R. Satchi-Fainaro, D. Shabat,
J. Am. Chem. Soc. 2016, 138, 13438-13446.
[32] S. Li, Q. Deng, Y. Zhang, X. Li, G. Wen, X. Cui, Y. Wan, Y. Huang, J.
Chen, Z. Liu, L. Wang, C. S. Lee, Adv. Mater. 2020, 32, 2001146.
[33] G. Ferrer-Sueta, N. Campolo, M. Trujillo, S. Bartesaghi, S. n. Car-ballal,
N. Romero, B. Alvarez, R. Radi, Chem. Rev. 2018, 118, 1338-1408.
[34] K. Pu, A. J. Shuhendler, J. Rao., Angew. Chem. Int. Ed. 2013, 52,
10325–10329; Angew. Chem. 2013, 125, 10515-10519.
[35] M. L. Odyniec, S. J. Park, J. E. Gardiner, E. C. Webb, A. C. Sedgwick, J.
Yoon, S. D. Bull, H. M. Kim, T. D James, Chem. Sci. 2020, 11, 7329-
7334.
[4]
[5]
a) V. S. Lin, W. Chen, M. Xian, C. J. Chang, Chem. Soc. Rev. 2015, 44,
4596-4618; b) S. Beck, H. Koster, Anal. Chem. 1990, 62, 2258-2270; c)
A. V. Trofimov, R. F. Vasil'ev, K. Mielke, W. Adam, Photochem. Photobiol.
1995, 62, 35-43.
a) Q. Miao, C. Xie, X. Zhen, Y. Lyu, H. Duan, X. Liu, J. V. Jokerst, K. Pu,
Nat. Biotechnol. 2017, 35, 1102-1110; b) Y. Lyu, D. Cui, J. Huang, W.
Fan, Y. Miao, K. Pu, Angew. Chem. Int. Ed. 2019, 58, 4983-4987; Angew
Chem. 2019, 131, 5037-5041.
[6]
[7]
S. K. Sun, H. F. Wang, X. P. Yan, Acc. Chem. Res. 2018, 51, 1131-1143.
M. Vacher,; I. Fdez. Galván, B. W. Ding, S. Schramm, R. Berraud-Pache,
P. Naumov, N. Ferré, Y.J. Liu, I. Navizet, D. Roca-Sanjuán, W. J. Baader,
R. Lindh, Chem. Rev. 2018, 118, 6927-6974.
[36] J. Cao, W. An, A. G. Reeves, A. R. Lippert, Chem. Sci. 2018, 9, 2552-
2558.
[37] D. Asanuma, M. Sakabe, M. Kamiya, K. Yamamoto, J. Hiratake, M.
Ogawa, N. Kosaka, P. L. Choyke, T. Nagano, H. Kobayashi, Nat.
Commun. 2015, 6, 6463.
[8]
[9]
Q. Miao, K. Pu, Adv. Mater. 2018, 30, 1801778.
J. Yan, S. Lee, A. Zhang, J. Yoon, Chem. Soc. Rev. 2018, 47, 6900-6916.
[38] K. Gu, Y. Xu, H. Li, Z. Guo, S. Zhu, S. Zhu, P. Shi, T. D. James, H. Tian,
W. H. Zhu, J. Am. Chem. Soc. 2016, 138, 5334-5340.
[39] a) J. Zhang, P. Cheng, K. Pu, Bioconjug. Chem. 2019, 30, 2089-2101; b)
X. Zhen, J. Zhang, J. Huang, C. Xie, Q. Miao, K. Pu. Angew. Chem. Int.
Ed., 2018, 57, 7804–7808; Angew. Chem. 2018, 130, 7930-7934.
[40] G. P. Dimri, X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E. E.
Medrano, M. Linskens, I. Rubelj, O. Pereira-Smith, Proc. Natl. Acad. Sci.
U. S. A. 1995, 92, 9363-9367.
[10] a) Y. Jiang, J. Huang, X. Zhen, Z. Zeng, J. Li, C. Xie, Q. Miao, J. Chen,
P. Chen, K. Pu, Nat. Commun. 2019, 10, 2064; b) Y. Lyu, D. Cui, J.
Huang, W. Fan, Y. Miao, K. Pu. Angew. Chem. Int. Ed., 2019, 58, 4983-
4987; Angew. Chem. 2019, 131, 5037-5041.
[11] M. Yang, J. Huang, J. Fan, J. Du, K. Pu, X. Peng, Chem. Soc. Rev. 2020,
49, DOI: 10.1039/D0CS00348D.
[12] N. Hananya, D. Shabat, Angew. Chem. Int. Ed. 2017, 56, 16454-16463;
Angew. Chem. 2017, 129, 16674-16683.
[13] J. Yang, W. Yin, R. Van, K. Yin, P. Wang, C. Zheng, B. Zhu, K. Ran, C.
Zhang, M. Kumar, Y. Shao, C. Ran, Nat. Commun. 2020, 11, 4052.
[14] a) N. Hananya, D. Shabat, ACS Cent. Sci. 2019, 5, 949-959; b) W. Adam,
I. Bronstein, B. Edwards, T. Engel, D. Reinhardt, F. W. Schneider, A. V.
Trofimov, R. F. Vasil'ev, J. Am. Chem. Soc. 1996, 118, 10400-10407; c)
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10.1002/anie.202013531
Angewandte Chemie International Edition
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Near-infrared chemiluminophores with modular structures amenable to construction of activatable molecular probes are synthesized,
which can turn-on their chemiluminescence by the biomarker of interest at a record long emission wavelength above 750 nm.
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