Detection and cellular imaging of human cancer enzyme using a turn-on, wavelength-shiftable, self-immolative profluorophore

Article abstract of DOI:10.1021/ja5030707

A frontier area in the development of activatable (turn-on) fluorescence-based probes is that concerned with rapid and selective stimulus triggering of probe activation so as to allow for biomarker identification and cellular imaging. The work here is concerned with a cloaked fluorophore composed of a reporter whose fluorescence is efficiently quenched by it being bound to an activatable trigger group through a novel self-immolative linker. Highly selective and rapid activation of the trigger group is achieved by chemical and enzymatic means that result in activated trigger group detachment from the self-immolative linker, with the latter subsequently cleaved from the reporter autonomously, thereby unmasking intense, red-shifted fluorescence emission. To achieve this success, we used a trimethyl-locked quinone propionic acid trigger group and an N-methyl-p-aminobenzyl alcohol self-immolative linker attached to the reporter. Delineated here are the synthesis and characterization of this cloaked fluorophore and the evaluation of its triggered turning on in the presence of an up-regulated enzyme in human cancer cells, NAD(P)H:quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC 1.6.99.2).

Full text of DOI:10.1021/ja5030707

Communication
pubs.acs.org/JACS
Open Access on 05/09/2015
Detection and Cellular Imaging of Human Cancer Enzyme Using a
Turn-On, Wavelength-Shiftable, Self-Immolative Profluorophore
Suraj U. Hettiarachchi, Bijeta Prasai, and Robin L. McCarley*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, United States
S
* Supporting Information
cells by reporter fluorescence that is initiated by intracellular
ABSTRACT: A frontier area in the development of
activatable (turn-on) fluorescence-based probes is that
concerned with rapid and selective stimulus triggering of
probe activation so as to allow for biomarker identification
and cellular imaging. The work here is concerned with a
cloaked fluorophore composed of a reporter whose
fluorescence is efficiently quenched by it being bound to
an activatable trigger group through a novel self-
immolative linker. Highly selective and rapid activation
of the trigger group is achieved by chemical and enzymatic
means that result in activated trigger group detachment
from the self-immolative linker, with the latter sub-
sequently cleaved from the reporter autonomously,
thereby unmasking intense, red-shifted fluorescence
emission. To achieve this success, we used a trimethyl-
locked quinone propionic acid trigger group and an N-
methyl-p-aminobenzyl alcohol self-immolative linker at-
tached to the reporter. Delineated here are the synthesis
and characterization of this cloaked fluorophore and the
evaluation of its triggered turning on in the presence of an
up-regulated enzyme in human cancer cells, NAD(P)-
H:quinone oxidoreductase-1 (NQO1, DT-diaphorase, EC
1.6.99.2).
enzyme action; only three distinct cancer-linked enzymes have
been targeted to date.5−7 Enzyme-initiated dequenching of
probe fluorescence has been used to improve the signal-to-
background value of the resulting reporter in diseased
(positive) cells in comparison to that of unactivated probe or
unaffected (negative) cells. Probe fluorescence is quenched via
fluorescence resonance energy transfer (FRET) or photo-
induced electron-transfer (PeT) mechanisms, and reporter
fluorescence comes about by enzyme-initiated disassociation of
the FRET quencher/donor pair upon enzyme binding7 or
cleavage of the quencher (trigger group) from the probe.5,6
However, modest signal-to-background (SBR)/positive-to-
negative (PNR) or tumor-to-background (TBR) ratios have
been reported.5,6
Ideal probe characteristics include rapid cell uptake and
highly selective activation to yield the reporter, low
fluorescence efficiency (Φprobe) of the unactivated probe but
high fluorescence efficiency of the reporter (Φreport), and high-
quality retention of the reporter inside the diseased cells.
Furthermore, the probe−reporter pair will exhibit large
probe
report
differences in energies of absorption maxima (λ
vs λ
)
abs
abs
probe
and emission maxima (λ
vs λerempiosrt), so significantly
emis
enhanced PNR/TBR values arise from the low absorptivity of
the probe at the reporter’s maximum excitation wavelength.
A highly promising approach that incorporates many of the
above desired probe/reporter qualities is one wherein the probe
is tripartite, being composed of a self-immolative linker8
between the enzyme substrate/trigger group and the reporter
moiety in the fluorescently silent probe. As a result of enzyme
activation, the trigger group is first removed, and then the self-
immolative linker is autonomously eliminated to yield the now
highly fluorescent reporter. It is anticipated that by the careful
selection of linker characteristics, the optical properties of the
probe can be made distinct from those of the reporter, and the
linker can be quickly cleaved from the reporter subsequent to
trigger group activation.
he success of the ever-growing field of in vivo and ex vivo
T
fluorescence imaging of diseased cells for diagnosis,
pathology, and treatment applications rests upon the existence
of disease-specific molecules whose spectroscopic signal is
readily differentiated from that of the background of
surrounding normal cells.1 Molecular probes whose cloaked
fluorescence reporter signal is turned on by endogenous
enzymes offer distinct advantages over always-on reporters, in
particular, higher signal-to-background (positive-to-negative)
values.2 Critical for cancer imaging is the development of
libraries of turn-on probes that will allow for the collection of
real-time information on the diseased tissue cell microenviron-
ment and the high-fidelity definition of diseased and healthy
tissue borders during fluorescence-assisted surgical resec-
tions.3−5
Unfortunately, the number of pro-fluorogenic probes that
can have their fluorescence output selectively and quickly
revealed by the presence of cancer-associated enzyme is
extremely small, as is the type of disease-associated activating
enzyme. Also, routes to improving the positive-to-negative
value are limited. There are three extant probes that passively
target cancer cells and rapidly (<30 min) identify the diseased
To that end, we developed probe 1,9 consisting of a
trimethyl-locked quinone propionic acid (Q3PA) trigger
group10 linked to a fluorescently masked naphthalimide
(reporter 2) by N-methyl-p-aminobenzyl alcohol, NMPABA.
The Q3PA trigger group was selected due to its known rapid
and highly selective reduction10 by NAD(P)H:quinone
oxidoreductase-1 (NQO1),11,12 an enzyme intimately associ-
ated with cancer13 and overexpressed 2- to 50-fold in the
Received: March 28, 2014
Published: May 9, 2014
© 2014 American Chemical Society
7575
dx.doi.org/10.1021/ja5030707 | J. Am. Chem. Soc. 2014, 136, 7575−7578

Journal of the American Chemical Society
Communication
the naphthalimide reporter by the Q3PA group. This
conclusion is supported by outcomes based on voltammetric
and spectroscopic data for probe 1, reporter 2, and Q3PA;9 the
free energy of the PeT process is −0.98 eV, indicating ready
electron transfer from the excited naphthalimide to the
electron-poor Q3PA group of probe 1. In comparison to the
previously reported probe Q3NI,5 there is a 3-fold-larger change
in fluorescence efficiency for probe 1 upon NQO1 action to
yield the corresponding reporter (Φreport/Φprobe), and the
fluorescence energy range of reporter 2 is more attractive for
possible imaging applications.
Scheme 1. Turn-On Self-Immolative Profluorophore 1
We next wanted to demonstrate rapid turning on of reporter
2 by the self-immolative cleavage of the NMPABA linker
subsequent to the reduction of probe 1. Dithionite reduction of
the Q3PA moiety is complete within 1 s, and the half-life of the
lactonization reaction is less than 2 min.16 Thus, sodium
dithionite was added to aqueous solutions of probe 1, and the
resulting fluorescence from reporter 2 was used to determine
the extent of probe 1 conversion to reporter 2 (Figure 2).
cytosol of numerous human tumor cells (e.g., colon, breast,
pancreas, and nonsmall cell lung).11 The previously unreported
NQO1-selective, turn-on fluorescence probe 1 relies on tuning
the push−pull internal charge transfer (ICT)14 of the
naphthalimide scaffold via its attachment to the self-immolative
NMPABA linker using an electron-withdrawing carbamate
connection. We show here that upon initiation of trigger group
removal from the tripartite probe, subsequent rapid cleavage of
linker occurs so as to afford reporter 2, which has distinct
spectral properties such that NQO1-positive cancer cells can be
imaged and identified with a positive-to-negative ratio of
∼500:1.
As seen in Figure 1, the spectroscopic properties of probe 1
and reporter 2 in buffered aqueous media are strikingly
Figure 2. Reduction-stimulated turning on of reporter 2. Time-course
fluorescence (λex = 432 nm, λemis = 540 nm) from 10 μM probe 1 (pH
7.40, 0.1 M phosphate buffer) after reduction by 16 μM sodium
dithionite. The inset contains time-dependent spectra of 2 μM probe 1
upon reduction. T = 25 °C.
Reporter 2 is quickly turned on (t1/2 ∼6 min) under
physiologically relevant conditions, pointing to the rapid self-
immolative cleavage of the N-methyl-p-aminobenzyl alcohol
and CO2 loss8 to yield reporter 2; this occurs because of the
electron-rich character of the N-methyl-p-aminobenzyl alcohol
vs that of the more typically encountered p-aminobenzyl
alcohol linker.8,17 Conversion was confirmed by mass
spectrometry analysis of probe 1 solutions treated with
dithionite.9 On the basis of the relatively short time for the
turn-on process, the use of probe 1 should allow for detecting
the presence of NQO1 enzyme activity.
Figure 1. Absorbance and emission spectra of 2 μM probe 1 and
reporter 2 in pH 7.40, 0.1 M phosphate buffer. T = 25 °C.
To evaluate the ability of human NAD(P)H:quinone
oxidoreductase-1 (hNQO1) to initiate the decloaking of
probe 1 and reveal free reporter 2 upon Q3PA trigger group
activation, we utilized the fluorescent product formation
technique.18 Under in vitro conditions, solutions of probe 1
(2−60 μM) incubated with hNQO1 (40 μg) and its cofactor
NADH (100 μM) exhibited steady increases in fluorescence,
suggesting a relatively fast rate of reporter 2 production; control
experiments with NADH alone did not yield significant changes
in fluorescence, a result in accord with that previously shown
for the Q3PA trigger group.10 In addition, the Q3PA trigger
group is stable to reduction/addition reactions in the presence
of glutathione and ascorbate as well as dithiothreitol.5
different, and these differences are attributed to the presence of
the quinone propionic acid trigger group (Q3PA), as well as the
carbamate connection between the N-methyl-p-aminobenzyl
alcohol, NMPABA, linker, and the naphthalimide fluorophore.
probe
The λarebpsort is red shifted roughly 50 nm in comparison to λ
abs
(432 nm vs 380 nm), as is λreempiosrt vs λepmroibse (532 nm vs 480 nm),
with both observations being in accord with the presence of the
electron-deficient carbamate14 in probe 1. Interestingly, the
absorptivity of probe 1 at the maximum absorption wavelength
of reporter 2 is ∼5 times lower than that for reporter 2. The
Φ
probe value of 0.002, which is 95 times less than Φreport = 0.19,9
is due to photoinduced electron transfer (PeT)15 quenching of
7576
dx.doi.org/10.1021/ja5030707 | J. Am. Chem. Soc. 2014, 136, 7575−7578

Journal of the American Chemical Society
Communication
Furthermore, hNQO2 plus its cofactor13 is ineffective at
releasing reporter 2. From the hNQO1 enzyme experiments,
the initial rate of reporter 2 formation, V (μmol min−1 mg
hNQO1−1), was calculated and then plotted as a function of
probe 1 concentration (Figure 3). Apparent kinetic parameters
amide-linked napthalimide reporter, Q3NI.5 Wide-field fluo-
rescence micrographs yielded similar results. Upon statistical
evaluation of the fluorescence intensities in confocal images of
hNQO1-positive (35 samples) and hNQO1-negative (29
samples) cells like those in Figure 4, a value of 11 was found
for the positive-to-negative ratio (PNR).9 Similarly, for wide-
field fluorescence images (33 positive samples/20 negative
samples), a PNR of 510 was observed.9 As a direct point for
comparison, a PNR of 23 was obtained for the Q3NI probe
using the same HT29(NQO1-positive)/H596(NQO1-nega-
tive) cells during wide-field microscopy imaging experiments.5
Thus, the PNR value of ∼500 obtained with probe 1/reporter 2
is unprecedented in the context of our previous work with
identical cell lines, and this value can be improved in wide-field
measurements by the use of a filter set (BCECF) that more
completely encompasses the emission profile of reporter 2. It is
clear that the outcomes discussed here come about from the
exceedingly large difference in fluorescence efficiency of
reporter 2 vs probe 1 (95-fold increase) that results from the
hNQO1-initiated removal of the trigger group quencher and
self-immolative linker, the very high fluorescence efficiency of
reporter 2, the significantly energy-shifted absorption maxima,
and the correspondingly lower molar absorptivity of probe 1 vs
reporter 2 at the excitation maximum of reporter 2. Also, the
energies over which probe 1/reporter 2 is excited/emits light
are shifted to the red by roughly 100 nm when compared to
those of the previously documented Q3NI probe,5 leading to a
significantly decreased background (negative) signal.
In summary, we have presented the synthesis, properties, and
biological evaluation of a novel turn-on fluorescent probe for
the cancer-linked enzyme NQO1. The discrimination of human
cells possessing NQO1, from those that do not, is achieved in a
highly selective fashion, which results from the dramatically
increased fluorescence efficiency of the red-shifted reporter 2
that is generated only upon hNQO1 action on the trigger
group of probe 1. We are now using the ICT-tunable probe 1
and its variants to develop methods, including multiphoton
imaging routes,5 that allow for the identification of hard-to-
detect NQO1-expressing cancerous tissues, as well as the
diversification of disease targets20,21 for the activation of new
trigger groups. With regard to longer-term impacts, enzyme-
activatable probes will play important roles as “intelligent”
imaging agents applied to the fluorescence-assisted resection of
cancerous tissues22 and efficacy studies of personalized
chemotherapy.23
Figure 3. Kinetics of human NQO1 (40 μg) with probe 1 (2−60 μM)
in pH 7.4, 0.1 M phosphate buffer. Values (n = 3) are the average
1
sample standard deviation. The curve is the best fit to the average data.
T = 25 °C.
were obtained by fitting the data in Figure 3 to Michaelis−
Menten kinetics,19 namely, the Michaelis constant (Km) = 10.4
1.0 μM, maximum velocity (Vmax) = 0.00225
0.00008
μmol min−1 mg hNQO1−1, catalytic constant (kcat) = 0.068
0.002 min−1, and catalytic efficiency (kcat/Km) = 6.52 0.67 ×
103 M−1 min−1. Also, there is no apparent inhibition of hNQO1
activity with the [probe 1] values studied. Thus, substantial
reporter 2 release occurs upon probe 1 interaction with
hNQO1 under physiologically relevant conditions.
To investigate the possibility of differentiating types of cancer
cells based on hNQO1 content (positive vs negative), we
exposed live hNQO1-positive and hNQO1-negative cells to
probe 1 under typical culture conditions (37 °C/CO2
atmosphere). Confocal fluorescence microscopy images of
hNQO1-positive colorectal cancer cells (HT29) incubated with
probe 1 indicated significant probe 1 uptake and activation,
resulting in intense intracellular reporter 2 production.
However, hNQO1-negative nonsmall cell lung cancer cells
(H596) treated with probe 1 revealed minimal signal (Figure
4), pointing to the intracellular stability of probe 1 in NQO1-
negative cells, similar to what we have observed for another
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis conditions and spectral properties of probe 1 and
reporter 2, voltammetric data, and PNR data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
tunnel@lsu.edu
Notes
The authors declare no competing financial interest.
Figure 4. Microscopy images of hNQO1-positive HT29 colon (A, B,
C) and hNQO1-negative H596 lung (D, E, F) cancer cells after
incubation at 37 °C with 20 μM probe 1. Confocal images in A, B, D,
and E; differential interference contrast in C and F. DRAQ5 nuclear
stain was used for B and E. Scale bar = 20 μm.
ACKNOWLEDGMENTS
■
This material is based upon work supported by the U.S.
National Institutes of Health (5R21CA135585, P42ES013648)
7577
dx.doi.org/10.1021/ja5030707 | J. Am. Chem. Soc. 2014, 136, 7575−7578

Journal of the American Chemical Society
Communication
and the U.S. National Science Foundation under grant CHE-
0910845.
REFERENCES
■
(1) Frangioni, J. V. J. Clin. Oncology 2008, 26, 4012−4021.
(2) Kobayashi, H.; Choyke, P. L. Acc. Chem. Res. 2011, 44, 83−90.
(3) Nguyen, Q. T.; Olson, E. S.; Aguilera, T. A.; Jiang, T.; Scadeng,
M.; Ellies, L. G.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2010, 107,
4317−4322.
(4) Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. Biomaterials 2011,
32, 7127−7138.
(5) Silvers, W. C.; Prasai, B.; Burk, D. H.; Brown, M. L.; McCarley, R.
L. J. Am. Chem. Soc. 2013, 135, 309−314.
(6) Verdoes, M.; Oresic Bender, K.; Segal, E.; van der Linden, W. A.;
Syed, S.; Withana, N. P.; Sanman, L. E.; Bogyo, M. J. Am. Chem. Soc.
2013, 135, 14726−14730.
(7) Zhang, H.; Fan, J.; Wang, J.; Dou, B.; Zhou, F.; Cao, J.; Qu, J.;
Cao, Z.; Zhao, W.; Peng, X. J. Am. Chem. Soc. 2013, 135, 17469−
17475.
(8) Erez, R.; Shabat, D. Org. Biomol. Chem. 2008, 6, 2669−2672.
(9) See Supporting Information.
(10) Mendoza, M. F.; Hollabaugh, N. M.; Hettiarachchi, S. U.;
McCarley, R. L. Biochemistry 2012, 51, 8014−8026.
(11) Danson, S.; Ward, T. H.; Butler, J.; Ranson, M. Can. Treat. Rev.
2004, 30, 437−449.
(12) Ernster, L. Chem. Scr. 1987, 27A, 1−13.
(13) Siegel, D.; Yan, C.; Ross, D. Biochem. Pharmacol. 2012, 83,
1033−1040.
(14) Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J. J. Am.
Chem. Soc. 2008, 130, 4596−4597.
(15) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer: New York, 2006.
(16) Perera, I. Master’s Thesis. Louisiana State University, 2012.
(17) Robbins, J. S.; Schmid, K. M.; Phillips, S. T. J. Org. Chem. 2013,
78, 3159−3169.
(18) Beija, M.; Afonso, C. A.; Martinho, J. M. Chem. Soc. Rev. 2009,
38, 2410−2433.
(19) Cleland, W. W. Methods Enzymol. 1979, 63, 103−138.
(20) Li, L.; Zhang, C.-W.; Chen, G. Y. J.; Zhu, B.; Chai, C.; Xu, Q.-
H.; Tan, E.-K.; Zhu, Q.; Lim, K.-L.; Yao, S. Q. Nat. Commun. 2014, 5,
3276.
(21) Wang, R.; Chen, L.; Liu, P.; Zhang, Q.; Wang, Y. Chem.Eur. J.
2012, 18, 11343−11349.
(22) Choi, H. S.; Gibbs, S. L.; Lee, J. H.; Kim, S. H.; Ashitate, Y.; Liu,
F.; Hyun, H.; Park, G.; Xie, Y.; Bae, S.; Henary, M.; Frangioni, J. V.
Nat. Biotechnol. 2013, 31, 148−153.
(23) Kamiyama, H.; Rauenzahn, S.; Shim, J. S.; Karikari, C. A.;
Feldmann, G.; Hua, L.; Kamiyama, M.; Schuler, F. W.; Lin, M.-T.;
Beaty, R. M.; Karanam, B.; Liang, H.; Mullendore, M. E.; Mo, G.;
Hidalgo, M.; Jaffee, E.; Hruban, R. H.; Jinnah, H. A.; Roden, R. B. S.;
Jimeno, A.; Liu, J. O.; Maitra, A.; Eshleman, J. R. Clin. Cancer Res.
2013, 19, 1139−1146.
7578
dx.doi.org/10.1021/ja5030707 | J. Am. Chem. Soc. 2014, 136, 7575−7578