Norcyanine-Carbamates Are Versatile Near-Infrared Fluorogenic Probes

Article abstract of DOI:10.1021/jacs.1c02112

Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (～170×) due to dual requirements for NIR emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex in vivo settings, a γ-glutamate substituted CyBam was applied to imaging γ-glutamyl transpeptidase (GGT) activity in a metastatic model of ovarian cancer. Overall, CyBams have significant potential to extend the reach of fluorogenic strategies to intact tissue and live animal imaging applications.

Full text of DOI:10.1021/jacs.1c02112

pubs.acs.org/JACS
Communication
Norcyanine-Carbamates Are Versatile Near-Infrared Fluorogenic
Probes
Syed Muhammad Usama, Fuyuki Inagaki, Hisataka Kobayashi, and Martin J. Schnermann*
Cite This: J. Am. Chem. Soc. 2021, 143, 5674−5679
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in
living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly
used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate
linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (∼170×) due to dual requirements for NIR
emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex in vivo
settings, a γ-glutamate substituted CyBam was applied to imaging γ-glutamyl transpeptidase (GGT) activity in a metastatic model of
ovarian cancer. Overall, CyBams have significant potential to extend the reach of fluorogenic strategies to intact tissue and live
animal imaging applications.
electively activating a fluorescent signal is a powerful
approach to interrogate biological processes. A common
S
tactic uses multichromophore systems that rely on quenching
through fluorescence resonance energy transfer (FRET) and
related mechanisms.1 Another approach uses fluorogenic
probes, where the change in signal results from chemical
transformations to the chromophore itself.2−6 The latter often
provides improved turn-ON ratios and benefits from requiring
only a single chromophore. The most broadly used fluorogenic
chemistry is based on derivatization of coumarin, rhodamine,
and hybrid cyanine scaffolds, which absorb and emit light in
the visible to far-red range.4,5,7−10 To carry out such
experiments in living organisms, it is desirable to have turn-
ON probes that absorb and emit long-wavelength near-infrared
(NIR) light (>700 nm), which is less attenuated by tissue.
However, only systems based on FRET pairs or self-quenching
have routinely been applied in this range (Figure 1A).11−14
Indocyanine dyes are exceptionally useful fluorescent probes.
Heptamethine cyanines such as indocyanine green (ICG) and
IR-800CW have been the subject of extensive preclinical and
clinical in vivo imaging efforts.15,16 Fluorogenic probes built on
the heptamethine cyanine scaffold would benefit from the
extensive infrastructure built for their use, as well as previous
efforts to optimize these molecules.13,14 We hypothesized that
such probes could be created by carbamate derivatization of
norcyanines; a class of molecules originally reported by Miltsov
characterized by secondary, not tertiary, indolenine nitrogen
atoms attached to the polymethine chromophore.17−19 These
dyes differ from conventional cyanines in that their NIR
fluorescent signal requires nitrogen protonation (pKa ∼ 5).
Achilefu and co-workers have demonstrated the exceptional
utility of water-soluble variants of these dyes for in vivo tumor
imaging.20,21
Figure 1. (a) Previous NIR turn-ON approaches and (b) fluorogenic
CyBams reported here.
that sulfonated heptamethine norcyanines can be derivatized
with a range of cleavable benzyl carbamates to provide the
stable CyBam probes (λabs = 430 nm). These molecules then
undergo efficient triggered 1,6-elimination to provide the pH-
responsive heptamethine norcyanines (protonated form λabs
=
755 nm). Unlike existing far-red fluorogenic probes, the
cationic chromophore is only formed through carbamate
cleavage and nitrogen protonation, leading to an exceptional
turn-ON ratio (>150×). A glutamate substituted CyBam can
Received: February 23, 2021
Published: April 12, 2021
Here, we report the synthesis, validation, and application of
a series of fluorogenic cyanine carbamates, or CyBams, that can
be activated by a range of stimuli (Figure 1B). We demonstrate
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679
© 2021 American Chemical Society
5674

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
be activated by γ-glutamyl transpeptidase (GGT) with high
selectivity in vitro and in a metastatic model of ovarian cancer.
Overall, these studies provide a new class of versatile NIR
fluorogenic probes with significant potential to extend turn-
ON strategies to in vivo applications.
To test this approach, we first prepared a disulfonated
heptamethine norcyanine, NorCy7, a decarboxylated form of a
compound previously reported by Achilefu.21 In line with prior
reports, the dye exhibits two forms in biologically relevant
NIR range at either neutral (pH 7.2) or acidic (pH 4.5)
conditions (Figure 2B, Figure S2). As anticipated, examining
the absorbance profile of CyBam-N3 and NorCy7 at both
neutral and acidic pH revealed three readily distinguishable
species (Figure 2B,C). We investigated the stability of CyBam-
N3 at physiological pHs and in serum and observed little
degradation (<5%) over 24 h (Figure S4). Next, we examined
the fluorogenic response of CyBam-N3 by incubating with
PPh3 at pH 5.2 (Figure 2D). We observed rapid conversion to
NorCy7-[H+] with a dramatic 170-fold increase in the
fluorescence signal (Figure 2E). This reaction could also be
carried out at neutral pH to provide the neutral form NorCy7
(Figure S3). Lastly, we compared the magnitude of the turn-
ON response of CyBams to xanthene cyanines, which are far-
red probes that have been broadly employed for in vivo
fluorogenic imaging.10,25−28 We prepared and tested a
sulfonated, acetylated variant and found turn-ON ratios of
1.5 and 15 (with 640 nm ex.) at pH 4.5 and 7.2, respectively
(Figure S5). The critical distinction is the absorption profile in
the OFF state. The acetylated xanthene cyanines exhibit a
substantial long-wavelength absorption band, which leads to
significant emission from the quenched state (Figure S5). By
contrast, CyBams exhibit minimal absorption in the NIR
region at either neutral or acidic pHs.
conditions, an unprotonated quenched form, NorCy7 (λabs
=
520 nm @ pH 7.4), and a protonated fluorescent form,
NorCy7-[H+] (λabs = 755 nm @ pH 4.5), with a pKa of 5.2
(Figure S1a,b). After examining several conditions, we were
delighted to find that exposure of NorCy7 and 4-nitrophenyl
carbonates to NaH or Cs2CO3 in DMF afforded the
corresponding carbamate products. Using this approach, we
prepared CyBam-N3 from NorCy7 and 1 in reasonable yield
following purification by reversed-phase chromatography
(Scheme 1).
Scheme 1. Synthesis of CyBam-N3
To investigate the utility of CyBams for cellular and in vivo
imaging, we used a validated fluorogenic trigger with significant
translational potential. GGT is a cell-surface-bound enzyme
involved in maintaining cellular glutathione (GSH) and
cysteine homeostasis.29−32 Additionally, it has been used as a
biomarker of several malignant tumors (including liver,
cervical, and ovarian), and overexpression of GGT has been
correlated with metastases.33−36 The key cleavable glutamate
was installed on CyBam-γ-Glu (Figure 3A) using a similar
procedure to that described above (see the Supporting
Information). After confirming the stability of CyBam-γ-Glu
(Figure S6), we examined the probe in enzymatic assays. We
CyBam-N3 allowed us to examine the turn-ON chemistry
using a chemical trigger. Azide reduction, involving aza-ylide
formation and hydrolysis, was hypothesized to initiate 1,6-
elimination and carbamic acid hydrolysis to result in
unmasking of the pH-sensitive norcyanine (Figure 2A).22−24
CyBam-N3 exhibits minimal absorbance and emission in the
Figure 2. (a) The turn-ON mechanism of CyBam-N3 is a two-step process requiring (i) cleavage of the carbamate linker and (ii) indolenine
protonation. (b) Absorbance spectra and (c) images of 10 μM of solutions CyBam-N3 (blue) and NorCy7 (green) in PBS, pH 7.4, and NorCy7-
[H+] (red) in acetate buffer, pH 5.2. (d) CyBam-N3 (10 μM; 1 equiv) and PPh3 (100 μM; 10 equiv) in PBS:MeOH (1:1), pH 5.2, were monitored
at 5 min intervals (n = 3). Complete conversion to NorCy7-[H+] occurred within 60 min of PPh3 addition. (e) Conversion of CyBam-N3 (10 μM)
to NorCy7-[H+] (100 μM PPh3, pH 4.5) resulted in a 170× enhancement of the NIR fluorescent signal (ex. 710 nm).
5675
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
Figure 3. (a) Structures of CyBam-γ-Glu and CyBam-N.C. (b) Increase in fluorescent intensity of CyBam-γ-Glu (20 μM) after incubation with
increasing concentration of GGT (0−400 U/L). (c) Activation of CyBam-γ-Glu after incubation with GGT (100 U/L), leucine aminopeptidase
(LAP; 800 U/L), and pig-liver esterase (PLE; 800 U/L) at 37 °C for 30 min in PBS pH 7.4 followed by pH adjustment to pH 5.2. Cathepsin B
(CatB; 2.5 μg) was used in acetate buffer pH 5.2 for 30 min at 37 °C. (d) Confocal images of activation of CyBam-γ-Glu (20 μM) and CyBam-
N.C. (20 μM) in SHIN-3 cells. The fluorescent signal from the probe and nucleus (Hoechst) is shown in red and blue, respectively. Images were
taken using a 63× oil immersed lens (numerical aperture, N.A. 1.4). (e) Quantification of fluorescent signal after incubation of CyBam-γ-Glu (20
μM) in the presence of GGT inhibitors (DON, GGsTop) and CyBam-N.C. in SHIN-3 cells using flow cytometry. The geometric mean fluorescent
intensity ( SD) of the fluorescent signal in the cells is shown (n = 6 independent experiments). (f) In vivo imaging of the SHIN-3-ZsGreen
metastatic tumor model at 3 h. Green and red pseudocolors are used to represent the signal from the GFP and Cy7 channels, respectively.
Fluorescent line graph showing a correlation between the fluorescent signal from GFP and Cy7 channel across the metastatic tumor in two different
regions (A and B). Data points are displayed as mean SD, and the p-values were evaluated by the Student’s t-test (*** p-value ≤0.001).
observed a GGT-dependent increase in fluorescent signal
(Figure 3B) with a Michaelis constant (KM = 16 μM) similar
to those obtained with other GGT probes (Figure S7).37 We
also established that CyBam-γ-Glu is specifically activated by
GGT and did not show any significant signal when incubated
with representative proteases and esterases (Figure 3C). Lastly,
we determined the selectivity of CyBam-γ-Glu using
established GGT inhibitors.38,39 We observed a 60% and
80% decrease in fluorescent signal in the presence of DON and
GGsTop, respectively, confirming the selectivity of the probe
(Figure S8).
We then examined CyBam-γ-Glu, and the corresponding
noncleavable variant CyBam-N.C., in cellular assays and in vivo
imaging experiments (Figure 3A). We used a SHIN-3 ovarian
cancer cell line that has been previously shown to overexpress
GGT.40 We first established that CyBam-γ-Glu exhibited
minimal toxicity (Figure S9). We also established that NorCy7
exhibited significant cellular uptake in SHIN-3 cells. The
majority of the fluorescent signal was observed in lysosomes,
where the acidic microenvironment is likely responsible for
formation of NorCy7-[H+] (Figure S10).21 Next, we evaluated
the cellular activation and selectivity of CyBam-γ-Glu with and
without incubation with GGT inhibitors. Using confocal
microscopy and flow cytometry, we observed a strong
fluorescent signal in cells treated with CyBam-γ-Glu. By
contrast, minimal fluorescence signal was observed in cells that
were either treated with CyBam-N.C. or preincubated with
GGT inhibitors (Figure 3D,E and Figures S11 and S12).
Encouraged by these results, we tested CyBam-γ-Glu in a
metastatic tumor model of ovarian cancer. This model entails
intraperitoneal injection of SHIN-3-ZsGreen cells, resulting in
formation of a significant primary tumor in the greater
omentum and locally disseminated metastases.40 CyBam-γ-
Glu (30 nmol) was injected intraperitoneally in mice, which
were euthanized after 1, 3, and 6 h, and both the primary
tumor and local metastases were imaged. Excellent colocaliza-
tion between CyBam-γ-Glu and the ZsGreen signal suggests
that the probe was activated and taken up selectively by tumor
cells, with a significant signal at all three time points (Figure 3F
and Figures S13 and S14). We also confirmed that these
probes can be used in live mice using a conventional in vivo
imaging system (IVIS). To do this, we compared CyBam-γ-
Glu and CyBam-N.C. in an MDA-MB-468 xenograft, which is
a triple-negative breast cancer cell line with modest GGT
expression.41,42 The CyBam-γ-Glu can be readily visualized
with significant differences between the two agents in both
tumor and liver signals, as well as tumor-to-background ratios
(Figure S15).
Finally, to examine the versatility of this approach, we
prepared and initially characterized the utility of CyBam
5676
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
Authors
probes to visualize common reactive oxygen species (ROS)
with well-validated ROS-responsive triggers: boronic acid
Syed Muhammad Usama − Chemical Biology Laboratory,
Center for Cancer Research, National Cancer Institute,
Frederick, Maryland 21702, United States; orcid.org/
0000-0002-7487-1568
Fuyuki Inagaki − Molecular Imaging Branch, Center for
Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892, United
States
Hisataka Kobayashi − Molecular Imaging Branch, Center for
Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892, United
States; orcid.org/0000-0003-1019-4112
7,43,44
−
(H2O2) and phosphine oxide (O2 ).
We tested the
induction of ROS in PC-3 cells, a prostate cancer cell line, by
doxorubicin. Notably, doxorubicin absorbs in the visible region
(λmax = 480 nm) which can hamper the utility of conventional
visible light-absorbing fluorogenic probes.45,46 As expected, we
observed the generation of both superoxide and hydrogen
peroxide, with no interference from the addition of
doxorubicin (Figures S16 and S17). These results suggest
that these probes may have significant utility in the exploration
of ROS biology.
Fluorogenic probes are powerful tools with the potential to
noninvasively monitor enzymatic processes and other stimuli
in real-time in living organisms. Here, we report CyBams, the
first enzyme- or analyte-responsive fluorogenic probes based
on the heptamethine cyanine scaffold. These readily water-
soluble probes result from modification of the norcyanine
scaffold with a cleavable carbamate linker that is activated
through 1,6-elimination and chromophore protonation. This
combination results in turn-ON ratios that dramatically exceed
those found with existing far-red fluorogenic probes,
particularly in acidic conditions. The results presented above
suggest that CyBams have significant potential for use as
activatable probes for in vivo imaging. We hypothesize their
application may include optically guided surgical procedures
and note that the extensive optical instrumentation in place for
heptamethine cyanines makes this prospect more enticing.47
Going forward, as CyBams are more emissive upon
protonation in the lysosome, it is possible that efforts to
improve their lysosomal targeting may serve to increase their
signal intensity. Additionally, as cyanine fluorophores have
historically been most useful as bioconjugatable probes, we
anticipate that the utility of CyBams may be enhanced when
combined with active targeting. In this scenario, targetable
CyBams create the possibility to report on enzymatic activity at
only a specific cell type or location of interest. Efforts toward
these goals are ongoing and will be reported in due course.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02112
Notes
The authors declare the following competing financial
interest(s): S.M.U. and M.J.S. have applied for a patent
based on this work.
ACKNOWLEDGMENTS
■
This work was supported by the Intramural Research Program
of the National Institutes of Health (NIH), NCI-CCR. We
acknowledge Dr. James A. Kelley (National Cancer Institute)
for providing the high-resolution mass spectrometry analysis.
We thank Dr. Gary T. Pauly (National Cancer Institute) for
assisting with LC/MS and HPLC purification. We would also
like to thank Dr. Valentin Magidson, NCI-Optical Microscopy
laboratory, and Dr. Jeff Carrell (CCR-Frederick Flow
Cytometry Core Laboratory) for assisting with confocal
microscopy and flow cytometry, respectively. Finally, we
thank Nimit L. Patel, Lisa Riffle and Joseph D. Kalen (Small
Animal Imaging Program), and Chelsea Sanders and Simone
Difilippantonio (Laboratory Animal Sciences Program) for
assistance with the in vivo study.
ABBREVIATIONS
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02112.
■
■
sı
NIR, near-infrared; FRET, fluorescence resonance energy
transfer; CyBam, cyanine carbamate; ICG, indocyanine
green; LAP, leucine aminopeptidase; PLE, pig-liver esterase;
CatB, cathepsin B; PBS, phosphate buffer saline; GGT, γ-
glutamyl transpeptidase; γ-Glu, γ-glutamate; DON, 6-diazo-5-
oxo-L-norleucine; GGsTop, 2-amino-4[3-(carboxymethyl)-
phenyl](methyl)phosphono-butanoic acid; GSH, glutathione;
GFP, green fluorescent protein; Cy7, heptamethine; DMF,
dimethylformamide; NaH, sodium hydride; Cs2CO3, cesium
carbonate; abs, absorbance; ex, excitation; em, emission; N.A.,
numerical aperture
Synthetic details and characterization of CyBam-N3,
CyBam-γ-Glu, CyBam-N.C., CyBam-B(OH)2, CyBam-
P(OPh)2, and their intermediates; supplementary
figures; detailed information on enzymatic, in vitro, and
in vivo assays; additional data and figures including
absorbance and fluorescence spectra, photophysical
properties, Staudinger release, stability values, pKa
values, pH values, rate of activation, kinetics of
fluorogenic probe activation, inhibition quantification,
toxicity data, cellular uptake, confocal imaging, flow
cytometry analyses, and bright-field imaging (PDF)
REFERENCES
■
(1) Wu, L.; Huang, C.; Emery, B. P.; Sedgwick, A. C.; Bull, S. D.;
He, X. P.; Tian, H.; Yoon, J.; Sessler, J. L.; James, T. D. Forster
resonance energy transfer (FRET)-based small-molecule sensors and
imaging agents. Chem. Soc. Rev. 2020, 49 (15), 5110−5139.
(2) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y.
New strategies for fluorescent probe design in medical diagnostic
imaging. Chem. Rev. 2010, 110 (5), 2620−40.
(3) Grimm, J. B.; Heckman, L. M.; Lavis, L. D. The Chemistry of
Small-Molecule Fluorogenic Probes. Prog. Mol. Biol. Transl 2013, 113,
1−34.
AUTHOR INFORMATION
Corresponding Author
Martin J. Schnermann − Chemical Biology Laboratory,
Center for Cancer Research, National Cancer Institute,
Frederick, Maryland 21702, United States; orcid.org/
0000-0002-0503-0116; Email: martin.schnermann@
nih.gov
■
5677
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
(4) Chyan, W.; Raines, R. T. Enzyme-Activated Fluorogenic Probes
for Live-Cell and in Vivo Imaging. ACS Chem. Biol. 2018, 13 (7),
1810−1823.
(24) Wang, Y.; Li, J.; Feng, L.; Yu, J.; Zhang, Y.; Ye, D.; Chen, H. Y.
Lysosome-Targeting Fluorogenic Probe for Cathepsin B Imaging in
Living Cells. Anal. Chem. 2016, 88 (24), 12403−12410.
(25) Huang, J.; Lyu, Y.; Li, J.; Cheng, P.; Jiang, Y.; Pu, K. A Renal-
Clearable Duplex Optical Reporter for Real-Time Imaging of
Contrast-Induced Acute Kidney Injury. Angew. Chem., Int. Ed. 2019,
58 (49), 17796−17804.
(5) Jun, J. V.; Chenoweth, D. M.; Petersson, E. J. Rational design of
small molecule fluorescent probes for biological applications. Org.
Biomol. Chem. 2020, 18 (30), 5747−5763.
(6) Shieh, P.; Hangauer, M. J.; Bertozzi, C. R. Fluorogenic
azidofluoresceins for biological imaging. J. Am. Chem. Soc. 2012,
134 (42), 17428−31.
(26) Huang, J.; Li, J.; Lyu, Y.; Miao, Q.; Pu, K. Molecular optical
imaging probes for early diagnosis of drug-induced acute kidney
injury. Nat. Mater. 2019, 18 (10), 1133−1143.
(7) Chan, J.; Dodani, S. C.; Chang, C. J. Reaction-based small-
molecule fluorescent probes for chemoselective bioimaging. Nat.
Chem. 2012, 4 (12), 973−84.
(27) Cheng, P.; Chen, W.; Li, S.; He, S.; Miao, Q.; Pu, K. Fluoro-
Photoacoustic Polymeric Renal Reporter for Real-Time Dual Imaging
of Acute Kidney Injury. Adv. Mater. 2020, 32 (17), e1908530.
(28) Huang, J.; Jiang, Y.; Li, J.; He, S.; Huang, J.; Pu, K. A Renal-
Clearable Macromolecular Reporter for Near-Infrared Fluorescence
Imaging of Bladder Cancer. Angew. Chem., Int. Ed. 2020, 59 (11),
4415−4420.
(29) West, M. B.; Chen, Y.; Wickham, S.; Heroux, A.; Cahill, K.;
Hanigan, M. H.; Mooers, B. H. Novel insights into eukaryotic gamma-
glutamyltranspeptidase 1 from the crystal structure of the glutamate-
bound human enzyme. J. Biol. Chem. 2013, 288 (44), 31902−13.
(30) Griffith, O. W.; Meister, A. Translocation of intracellular
glutathione to membrane-bound gamma-glutamyl transpeptidase as a
discrete step in the gamma-glutamyl cycle: glutathionuria after
inhibition of transpeptidase. Proc. Natl. Acad. Sci. U. S. A. 1979, 76
(1), 268−72.
(31) Griffith, O. W.; Bridges, R. J.; Meister, A. Evidence that the
gamma-glutamyl cycle functions in vivo using intracellular gluta-
thione: effects of amino acids and selective inhibition of enzymes.
Proc. Natl. Acad. Sci. U. S. A. 1978, 75 (11), 5405−8.
(32) Wickham, S.; West, M. B.; Cook, P. F.; Hanigan, M. H.
Gamma-glutamyl compounds: substrate specificity of gamma-
glutamyl transpeptidase enzymes. Anal. Biochem. 2011, 414 (2),
208−14.
(33) Schafer, C.; Fels, C.; Brucke, M.; Holzhausen, H. J.; Bahn, H.;
Wellman, M.; Visvikis, A.; Fischer, P.; Rainov, N. G. Gamma-glutamyl
transferase expression in higher-grade astrocytic glioma. Acta Oncol.
2001, 40 (4), 529−35.
(34) Yao, D.; Jiang, D.; Huang, Z.; Lu, J.; Tao, Q.; Yu, Z.; Meng, X.
Abnormal expression of hepatoma specific gamma-glutamyl trans-
ferase and alteration of gamma-glutamyl transferase gene methylation
status in patients with hepatocellular carcinoma. Cancer 2000, 88 (4),
761−9.
(35) Luo, M.; Sun, W.; Wu, C.; Zhang, L.; Liu, D.; Li, W.; Mei, Q.;
Hu, G. High pretreatment serum gamma-glutamyl transpeptidase
predicts an inferior outcome in nasopharyngeal carcinoma. Oncotarget
2017, 8 (40), 67651−67662.
(36) Hanigan, M. H.; Frierson, H. F., Jr.; Brown, J. E.; Lovell, M. A.;
Taylor, P. T. Human ovarian tumors express gamma-glutamyl
transpeptidase. Cancer Res. 1994, 54 (1), 286−290.
(8) Zhang, J.; Chai, X.; He, X. P.; Kim, H. J.; Yoon, J.; Tian, H.
Fluorogenic probes for disease-relevant enzymes. Chem. Soc. Rev.
2019, 48 (2), 683−722.
(9) Karton-Lifshin, N.; Albertazzi, L.; Bendikov, M.; Baran, P. S.;
Shabat, D. ″Donor-two-acceptor″ dye design: a distinct gateway to
NIR fluorescence. J. Am. Chem. Soc. 2012, 134 (50), 20412−20.
(10) Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S.
A unique approach to development of near-infrared fluorescent
sensors for in vivo imaging. J. Am. Chem. Soc. 2012, 134 (32), 13510−
23.
(11) Widen, J. C.; Tholen, M.; Yim, J. J.; Antaris, A.; Casey, K. M.;
Rogalla, S.; Klaassen, A.; Sorger, J.; Bogyo, M. AND-gate contrast
agents for enhanced fluorescence-guided surgery. Nat. Biomed Eng.
2021, 5, 264.
(12) Ofori, L. O.; Withana, N. P.; Prestwood, T. R.; Verdoes, M.;
Brady, J. J.; Winslow, M. M.; Sorger, J.; Bogyo, M. Design of Protease
Activated Optical Contrast Agents That Exploit a Latent Lysosomo-
tropic Effect for Use in Fluorescence-Guided Surgery. ACS Chem.
Biol. 2015, 10, 1977.
(13) Chen, C.; Tian, R.; Zeng, Y.; Chu, C.; Liu, G. Activatable
Fluorescence Probes for ″Turn-On″ and Ratiometric Biosensing and
Bioimaging: From NIR-I to NIR-II. Bioconjugate Chem. 2020, 31 (2),
276−292.
(14) Gorka, A. P.; Nani, R. R.; Schnermann, M. J. Cyanine polyene
reactivity: scope and biomedical applications. Org. Biomol. Chem.
2015, 13 (28), 7584−98.
(15) Gorka, A. P.; Nani, R. R.; Schnermann, M. J. Harnessing
Cyanine Reactivity for Optical Imaging and Drug Delivery. Acc. Chem.
Res. 2018, 51 (12), 3226−3235.
(16) Usama, S. M.; Thapaliya, E. R.; Luciano, M. P.; Schnermann,
M. J. Not so innocent: Impact of fluorophore chemistry on the in vivo
properties of bioconjugates. Curr. Opin. Chem. Biol. 2021, 63, 38−45.
(17) Miltsov, S.; Encinas, C.; Alonso, J. N. Nortricarbocyanines:
New near-infrared pH-indicators. Tetrahedron Lett. 1998, 39 (50),
9253−9254.
(18) Briggs, M. S.; Burns, D. D.; Cooper, M. E.; Gregory, S. J. A pH
sensitive fluorescent cyanine dye for biological applications. Chem.
Commun. 2000, No. 23, 2323−2324.
(19) Zhang, Z.; Achilefu, S. Design, synthesis and evaluation of near-
infrared fluorescent pH indicators in a physiologically relevant range.
Chem. Commun. 2005, No. 47, 5887−9.
(20) Lee, H.; Akers, W.; Bhushan, K.; Bloch, S.; Sudlow, G.; Tang,
R.; Achilefu, S. Near-infrared pH-activatable fluorescent probes for
imaging primary and metastatic breast tumors. Bioconjugate Chem.
2011, 22 (4), 777−84.
(21) Gilson, R. C.; Tang, R.; Som, A.; Klajer, C.; Sarder, P.; Sudlow,
G. P.; Akers, W. J.; Achilefu, S. Protonation and Trapping of a Small
pH-Sensitive Near-Infrared Fluorescent Molecule in the Acidic
Tumor Environment Delineate Diverse Tumors in Vivo. Mol.
Pharmaceutics 2015, 12 (12), 4237−46.
(22) Lukasak, B.; Morihiro, K.; Deiters, A. Aryl Azides as Phosphine-
Activated Switches for Small Molecule Function. Sci. Rep. 2019, 9 (1),
1470.
(37) Li, L.; Shi, W.; Wu, X.; Li, X.; Ma, H. In vivo tumor imaging by
a gamma-glutamyl transpeptidase-activatable near-infrared fluorescent
probe. Anal. Bioanal. Chem. 2018, 410 (26), 6771−6777.
(38) Han, L.; Hiratake, J.; Kamiyama, A.; Sakata, K. Design,
synthesis, and evaluation of gamma-phosphono diester analogues of
glutamate as highly potent inhibitors and active site probes of gamma-
glutamyl transpeptidase. Biochemistry 2007, 46 (5), 1432−47.
(39) Terzyan, S. S.; Cook, P. F.; Heroux, A.; Hanigan, M. H.
Structure of 6-diazo-5-oxo-norleucine-bound human gamma-glutamyl
transpeptidase 1, a novel mechanism of inactivation. Protein Sci. 2017,
26 (6), 1196−1205.
(40) Urano, Y.; Sakabe, M.; Kosaka, N.; Ogawa, M.; Mitsunaga, M.;
Asanuma, D.; Kamiya, M.; Young, M. R.; Nagano, T.; Choyke, P. L.;
Kobayashi, H. Rapid cancer detection by topically spraying a gamma-
glutamyltranspeptidase-activated fluorescent probe. Sci. Transl. Med.
2011, 3 (110), 110ra119.
(23) Hai, Z.; Wu, J.; Wang, L.; Xu, J.; Zhang, H.; Liang, G.
Bioluminescence Sensing of gamma-Glutamyltranspeptidase Activity
In Vitro and In Vivo. Anal. Chem. 2017, 89 (13), 7017−7021.
(41) Pompella, A.; De Tata, V.; Paolicchi, A.; Zunino, F. Expression
of gamma-glutamyltransferase in cancer cells and its significance in
drug resistance. Biochem. Pharmacol. 2006, 71 (3), 231−8.
5678
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679

Journal of the American Chemical Society
pubs.acs.org/JACS
Communication
(42) Dawson, J.; Smith, G. D.; Boak, J.; Peters, T. J. gamma-
Glutamyltransferase in human and mouse breast tumours. Clin. Chim.
Acta 1979, 96 (1−2), 37−42.
(43) Xu, K.; Liu, X.; Tang, B.; Yang, G.; Yang, Y.; An, L. Design of a
phosphinate-based fluorescent probe for superoxide detection in
mouse peritoneal macrophages. Chem. - Eur. J. 2007, 13 (5), 1411−6.
(44) Chang, M. C.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. A selective,
cell-permeable optical probe for hydrogen peroxide in living cells. J.
Am. Chem. Soc. 2004, 126 (47), 15392−3.
(45) Wagner, B. A.; Evig, C. B.; Reszka, K. J.; Buettner, G. R.; Burns,
C. P. Doxorubicin increases intracellular hydrogen peroxide in PC3
prostate cancer cells. Arch. Biochem. Biophys. 2005, 440 (2), 181−90.
(46) Wellington, K. W. Understanding cancer and the anticancer
activities of naphthoquinones - a review. RSC Adv. 2015, 5 (5),
20309−20338.
(47) Barth, C. W.; Gibbs, S. L. Fluorescence Image-Guided Surgery -
a Perspective on Contrast Agent Development. Proc. SPIE Int. Soc.
Opt Eng. 2020, 11222, 18.
5679
https://doi.org/10.1021/jacs.1c02112
J. Am. Chem. Soc. 2021, 143, 5674−5679