Remarkable Enhancement of Chemiluminescent Signal by Dioxetane-Fluorophore Conjugates: Turn-ON Chemiluminescence Probes with Color Modulation for Sensing and Imaging

Article abstract of DOI:10.1021/jacs.6b09173

Chemiluminescence is among the most sensitive methods for achieving a high signal-to-noise ratio in various chemical and biological applications. We have developed a modular practical synthetic route for preparation of turn-ON fluorophore-tethered dioxeta

Full text of DOI:10.1021/jacs.6b09173

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Article
Remarkable Enhancement of Chemiluminescent Signal by
Dioxetane-Fluorophore Conjugates: Turn-ON Chemiluminescence
Probes with Color Modulation for Sensing and Imaging
Nir Hananya, Anat Eldar Boock, Christoph R. Bauer, Ronit Satchi-Fainaro, and Doron Shabat
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b09173 • Publication Date (Web): 21 Sep 2016
Downloaded from http://pubs.acs.org on September 21, 2016
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Remarkable Enhancement of Chemiluminescent Signal by Dioxetane-Fluorophore
Conjugates: Turn-ON Chemiluminescence Probes with Color Modulation for
Sensing and Imaging
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Nir Hananyaa, Anat Eldar Boockb, Christoph R. Bauerc, Ronit Satchi-Fainarob, and
Doron Shabata*
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aSchool of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences and
bDepartment of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv
c
University, Tel Aviv 69978 Israel. Bioimaging Center, University of Geneva, Geneva,
Switzerland.
*Corresponding Author:
Doron Shabat
Department of Organic Chemistry
School of Chemistry, Faculty of Exact Sciences
Tel Aviv University
Tel Aviv 69978, Israel
Tel: +972 (0) 3 640 8340
Fax: +972 (0) 3 640 9293
E-mail: chdoron@post.tau.ac.il
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Abstract: Chemiluminescence is among the most sensitive methods for achieving a
high signal-to-noise ratio in various chemical and biological applications. We have
developed a modular practical synthetic route for preparation of turn-ON
fluorophore-tethered dioxetane chemiluminescent probes. The chemiluminescent
emission of the probes was significantly amplified through energy transfer
mechanism under physiological conditions. Two probes were composed with green
and near-infrared (NIR) fluorescent dyes, tethered to Schaap's dioxetane. While both
probes were able to provide chemiluminescence in vivo images following
subcutaneous injection, only the NIR probe could provide chemiluminescence image
following intraperitoneal injection. These are the first in vivo images produced by
Schaap's dioxetane-chemiluminescence probes with no need of enhancer. So far,
chemiluminescence cell-images, could only be obtained with a luciferin-based probe.
Our NIR probe was able to image cells, transfected with β-galactosidase gene, by
chemiluminescence microscopy. We also report, for the first time, the instability of
dioxetane-fluorophore conjugates to ambient light. Our synthetic route effectively
overcomes this limitation through a late stage functionalization of the dioxetane
intermediate. We anticipate that our practical synthetic methodology will be useful
for preparation of various chemiluminescent probes for numerous applications.
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Introduction
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Chemiluminescence assays are widely utilized in various chemical and biological
applications due to their sensitivity and high signal-to-noise ratio.1,2 Unlike
fluorescence-based assays, in chemiluminescence no light excitation is required.
Therefore, background signal arising from autofluorescence does not exist when
chemiluminescence is used. Such circumstance makes chemiluminescence especially
useful for tissue and whole-body imaging.3-6
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Most of the chemiluminescent compounds, currently in use, are activated by
oxidation; i.e., a stable precursor is oxidized usually by hydrogen peroxide, to form
an oxidized high-energy intermediate, which then decomposes to generate an
excited species. The latter decays to its ground state by either light emission or by
energy transfer. Common probes that act on such chemiluminescence mechanism
are usually based on luminol7 and oxalate esters.8 Utilizing this oxidation-activated
chemiluminescence mode-of-action, several systems were developed for the in vivo
imaging of reactive oxygen species (ROS).9-16 Innately, chemiluminescence that is
exclusively activated by oxidation is limited for the detection and imaging of ROS.
However, in 1987 Paul Schaap developed a new class of chemiluminescent probes,
which can be activated by an enzyme or an analyte of choice.17-19 Schaap's
chemiluminescent probes are based on adamantylidene–dioxetane, which is
triggered through removal of a protecting group from a phenol (Figure 1). The
resulting phenolate disassembles via an intramolecular chemically initiated electron
exchange luminescence (CIEEL) mechanism to emit blue light.
*
O
O
MeO
MeO
O
O
O
O
O
O
PG
triggering
event
O
+
OMe
O
O
hv 470nm
+
OMe
Figure 1. Chemiluminescent activation pathway of Schaap’s adamantylidene–dioxetane.
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In bioassays, under aqueous conditions, Schaap's dioxetanes suffer from one major
limitation; their chemiluminescence efficiency decreases significantly through non-
radiative energy transfer processes (quenching) by interaction with water
molecules.20 A common way to amplify the chemiluminescence signal of Schaap's
dioxetanes is achieved through energy transfer from the resulting excited species
(the benzoate shown in Figure 1) to a nearby acceptor, which is a highly emissive
fluorophore under aqueous conditions.21,22 Therefore, a surfactant-dye adduct is
usually added in commercial chemiluminescent immunoassays. The surfactant
reduces water-induced quenching by providing a hydrophobic environment for the
excited chemiluminescent probe, which transfers its energy to excite a nearby
fluorogenic dye. Consequently, the low-efficiency luminescence process is amplified
up to 400-fold in aqueous medium.23 However, since the surfactant mode-of-action
relies on micelles formation, its functional concentration is relatively high (above the
critical micelle concentration - CMC).24 As micellar structures are not maintained
when animals are treated systemically, the surfactant-dye adduct approach is not
practical for in vivo detection or imaging of biological activity generated by enzymes
or chemical analytes.25
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To overcome the limitation of a two-component system (a dioxetane probe and a
surfactant-fluorescent dye adduct), a single component, comprised of dioxetane
conjugated with fluorophore is required. Two previous reports have described
synthesis of dioxetane-fluorogenic dye conjugates, in which the dioxetane effectively
transfers chemiluminescence energy to the tethered fluorophore to emit light at a
wavelength that can be varied by choice of fluorophore.26,27 In addition to signal
amplification, tethering of dioxetane with fluorophore also allows color modulation
and red-shifting of the emitted light; a significant requirement for bioimaging
applications.28-33 Herein we report the further elaboration of this approach by
developing more practical synthetic route that solves synthetic restrictions, for
preparation of dioxetane-fluorophore conjugates. We also report the preparation of
the first near-infrared (NIR) Schaap's dioxetane-fluorophore conjugate and
demonstrate the applicability of our approach for making chemiluminescence probes
suitable for in vivo whole-body imaging.
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Results and Discussion
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Design and synthesis of fluorophore-tethered dioxetane probes:
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The general structure of the dioxetane-fluorophore conjugate and its activation
mechanism are presented in Figure 2. Removal of the trigger by the analyte of
interest initiates the CIEEL mechanism, which leads to energy transfer from the
excited benzoate to the dye, resulting in excitation of the fluorophore. Thus, light
emission should occur from a highly emissive species (the fluorophore) at its
corresponding wavelength.
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O
O
MeO
MeO
O
O
triggering
event
Trigger
O
O
CIEEL
Fluorophore
Fluorophore
O
O
*
O
O
O
O
OMe
OMe
OMe
hv
+
ET
*
Fluorophore
Fluorophore
Fluorophore
Figure 2. Chemiluminescence activation pathway of dioxetane fluorophore conjugate.
We sought to develop a practical synthetic pathway that allows modular attachment
of a fluorophore to the phenolic ring. Dioxetane is usually prepared by reaction of
singlet oxygen with a double bond. Since conditions for production of singlet oxygen
are not always compatible with the presence of a fluorophore, we developed a late-
stage functionalization chemistry that allows attachment of the fluorophore after
preparation of the dioxetane. The synthesis of a dioxetane-fluorophore conjugate
designed for activation by β-galactosidase (as a model enzyme) is shown in Figure 3.
Commercially available aldehyde 1a was protected with trimethyl-orthoformate to
give acetal 1b, followed by additional protection of the phenol group with t-
butyldimethylsilyl (TBS) chloride to afford compound 1c. The latter was reacted with
trimethylphosphite to produce phosphonate 1d, which was condensed with 2-
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adamantanone via the Wittig-Horner reaction to give enol ether 1e. Deprotection of
the TBS group of 1e gave phenol 1f, which was alkylated with a bromo-galactose
derivative to afford compound 1g. Hartwig-Miyaura C-H borylation34 of 1g afforded
phenylboronic ester 1h, which was coupled with benzylbromide 1i via Suzuki
coupling reaction to give compound 1j. Oxidation of 1j with singlet oxygen gave an
N-hydroxy-succinimide (NHS)-ester functionalized dioxetane 1k. This NHS-ester
readily reacts with various amine-functionalized dyes to afford dioxetane
fluorophore conjugates 1m.
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Cl
O
Cl
O
Cl
O
TBSO
HO
TBSCl, imidazole
DCM, RT
CH(OCH3)3, Bu4NBr3
MeOH, RT
O
O
HO
H
94%
1c
95%
1b
1a
Cl
O
Cl
O
Cl
O
TBAF
THF
TBSO
O
P(OCH3)3, TiCl4
DCM, 0°
1. LDA, THF, -78°
2. 2-adamantanone
HO
TBSO
O
P
95%
O
85%
92%
1e
1f
1d
OAc
bis(pinacolato)diboron
[Ir(OMe)(1,5-cod)]2, BBBPY
THF, 80°C
OAc
AcO
AcO
AcO
AcO
Acetobromo-α-D-galactose
Cl
O
Cl
B
O
O
Ag2CO3
O
O
O
quinoline : DCM (2:1)
87%
OAc
OAc
89%
1h
1g
O
O
O
OAc
AcO
O
O
OAc
N
AcO
AcO
Cl
O
Cl
O
O
O
O
O
O
O
O
AcO
1i
OAc
OAc
Br
O2, methylene blue, hv
Pd(PPh3)4, K2CO3
dioxane, 110°C
DCM, RT
O
1k
O
1j
O
92%
O
O
N
N
40%
O
O
O
OH
HO
Cl
O
O
O
O
O
1. Fluorophore
Et3N, DMF
NH2
HO
OH
2. K2CO3, MeOH
1m
H
N
Fluorophore
O
Figure 3. Synthetic strategy for dioxetane fluorophore conjugates.
Following the synthetic strategy presented in Figure 3, we prepared three different
chemiluminescent probes for monitoring of β-galactosidase activity. The chemical
structures of the probes are shown in Figure 4. Probes 2 and 3 are composed of
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dioxetane tethered with the fluorogenic dyes fluorescein and quinone-cyanine35,36
(QCy), respectively. Probe 1 is a basic Schaap-dioxetane without a tethered dye. The
chlorine substituent on the phenolic ring was introduced in order to decrease the
pKa of the released phenol after cleavage of the β-galactosidase substrate. Such pKa
should allow the chemiexcitation pathway of the dioxetane to occur under
physiological conditions.
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OH
OH
HO
HO
HO
HO
Cl
Cl
O
O
O
O
O
O
O
O
O
O
OH
OH
CF3CO2
1
N
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O
HN
OH
HO
HO
N
Cl
H
O
O
O
O
O
OH
O
3
OH
O
O
N
S
HN
2
O
N
N
CO2H
H
H
Figure 4. Molecular structures of chemiluminescent probes with emission of three different
colors for monitoring of β-galactosidase activity.
Light-induced decomposition of dioxetane-dye conjugates:
While working on the synthesis of the dioxetane-fluorophore conjugates, we
encountered an unexpected phenomenon. Although probe 1 was seemed to be
photo-stable, probes 2 and 3 appeared to decompose under normal room
illumination conditions. We measured the photo-stability of the probes in aqueous
solution (PBS, pH 7.4) under normal room-illumination. The light-induced
decomposition was monitored over several hours by a reverse-phase HPLC assay
(Figure 5).
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100
80
60
40
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0
Probe 1
Probe 2
Probe 3
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Time (Hours)
Figure 5. Decomposition of probes 1, 2 and 3 under normal room-illumination conditions.
Probes (300 μM) were incubated in PBS [100 mM], pH 7.4 at ambient temperature.
Probe 1 was unaffected by light over a 12-hours period. Probe 3 exhibited
significantly higher photo-stability than probe 2. The light-induced decomposition
half-life of probe 2 was 45 minutes, whereas the half-life of probe 3 was about 6
hours. No decomposition of any of the probes was observed when the solutions
were kept in the dark. A possible mechanism for light-induced decomposition of the
dioxetane-fluorophore conjugates, might involve electron transfer from the excited
dye to the peroxy-dioxetane bond.48 Figure 6 illustrates a possible light-induced
decomposition mechanism. The fluorophore of conjugate I is excited by visible light
to form excited species II. Electron transfer from the LUMO orbital of the excited
fluorophore to the antibonding σ* orbital of O–O peroxide bond results in bond
cleavage and subsequent decomposition of the dioxetane into benzoate III and
adamantanone.
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LUMO
6
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σ*
Visible
Light
E
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HOMO
σ
Fluorophore
Dioxetane
O
O
MeO
MeO
O
O
Trigger
O
Trigger
O
hν
ET
I
II
Fluorophore
Fluorophore
*
O
Trigger
O
OMe
O
decomposition
III
Fluorophore
Figure 6. Proposed pathway for visible light-induced decomposition of dioxetane-
fluorophore conjugates.
The observed light instability of probes 2 and 3 underlines the advantage of our late-
stage functionalization strategy over previous reported synthetic methods for
dioxetane-fluorophore conjugates. The oxidation of the enol-ether to the dioxetane
is usually performed by singlet oxygen generated from oxygen by a light source and a
photosensitizer. Such conditions, if applied after the conjugation of the fluorophore,
could lead to decomposition of the dioxetane. We were able to avoid light-induced
decomposition by using late-stage functionalization chemistry that allows
attachment of the fluorophore only after preparation of the dioxetane.
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Energy transfer observed for dioxetane-dye conjugate vs. a mixture of dioxetane
and a dye:
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We first wanted to evaluate the efficiency of energy transfer in the dioxetane-
fluorophore conjugates in comparison to a 1:1 mixture of probe 1 and a dye, upon
activation with β-galactosidase. The obtained chemiluminescence emission spectra
are shown in Figure 7. In the case of fluorescein, two emission maxima were
obtained for the dioxetane-dye mixture (Figure 7A) at wavelengths of 470 nm and
535 nm. These wavelengths correspond to the direct chemiluminescence (DCL) of
probe 1 (see ESI) and to the emission of fluorescein resulting from energy transfer
(ET), respectively. On the other hand, probe 2 (dioxetane-fluorescein conjugate),
upon activation by β-galactosidase, decomposed to emit greenish light with
maximum emission wavelength of 535 nm exclusively (Figure 7B). The observed
chemiluminescence spectrum of the probe was almost identical to its fluorescence
spectrum (dotted line); indicating a complete energy transfer to the fluorescein
acceptor. In the case of QCy, only blue emission with maximum wavelength of 470
nm was obtained for the dioxetane-dye mixture (Figure 7C). This emission
corresponds to the direct chemiluminescence of probe 1. On the other hand, probe
3 (QCy-tethered dioxetane) decomposed to emit NIR light with maximum emission
wavelength of 714 nm (Figure 7D). Similarly, as observed for probe 2, the
chemiluminescence spectrum of probe 3 was found to be almost identical to its
fluorescence spectrum (dotted line). This observation clearly supports the energy
transfer mechanism illustrated in Figure 2, and properly demonstrates the
significance of covalent conjugation between the dioxetane and the dye.
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Figure 7. Chemiluminescence emission spectra of 1μM (A) 1:1 mixture of probe 1 and
fluorescein derivative 2b (see ESI), λmax=470 nm, 535 nm; (B) Probe 2, λmax=535 nm; (C)
1:1 mixture of probe 1 and QCy derivative 3b (See ESI), λmax=470 nm; (D) Probe 3,
λmax=714 nm. Spectra were recorded in PBS [100 mM], pH 7.4 in the presence of 1.5
units/mL β-galactosidase (continuous lines). Fluorescence emission spectra (dotted lines).
Chemiluminescence parameters measured for probes 1, 2 and 3 and their ability to
detect and image β-galactosidase:
Next, we measured the light emission of the probes, as a function of time, in the
presence and in the absence of β-galactosidase. The probes exhibited a typical
chemiluminescent kinetic profile in the presence β-galactosidase with an initial signal
increase to a maximum followed by a slow decrease to zero (Figure 8 A, B and C). No
light emission was observed from the probes in the absence of β-galactosidase.
Figures 8D, 8E and 8F show the total photon counts emitted from each of the
probes. The measured chemiluminescence parameters obtained for probes 1, 2 and
3 are summarized in Table 1.
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Figure 8. (A-C) Chemiluminescent kinetic profiles of 1 μM (A) Probe 1, (B) Probe 2, and (C)
Probe 3 in PBS [100 mM], pH 7.4 in the presence of 1.5 units/mL β-galactosidase (blue,
yellow and grey lines, respectively) and in the absence of β-galactosidase (red line). (D-F)
Total photon counts emitted from (D) Probe 1, (E) Probe 2 and (F) Probe 3 in the presence of
β-galactosidase.
Table 1. Chemiluminescent parameters obtained for probes 1, 2 and 3.
λ max (nm)
470
T1/2 (min)
40
φ CL
Relative φ CL
Probe 1
Probe 2
Probe 3
3.3×10-5
3.8×10-3
9.0×10-4
1
535
170
114
27.2
714
100
The chemiluminescence quantum yield (φ CL) of probes 2 and 3 was calculated using
that of probe 1 as a known standard.37 Probes 2 and 3 exhibited significantly higher
light emission than the emission exhibited by probe 1 (114-fold for probe 2 and 27-
fold for probe 3). In addition, probes 2 and 3 had longer half-lives of light emission
under identical conditions.
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Probe 2 exhibited brighter chemiluminescence than the other probes since its
energy transfer is resulted with an excited fluorescein species (a dye with 90%
fluorescence quantum yield). We therefore selected probe 2 and demonstrated its
ability to detect β-galactosidase (Figure 9). The probe was incubated with different
concentrations of β-galactosidase and total chemiluminescence emission was
collected over 1 hour period. Linear correlation was observed between enzyme
concentrations and integrated chemiluminescence signal, enabling quantification of
enzyme concentration. We determined a detection limit (blank control + 3 S.D.) of
4.0x10-3.
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Figure 9. Total light emitted from 10 µM probe 2 in PBS [100 mM], pH 7.4 over a period of 1
hour, with different concentrations of β-galactosidase. The inset focuses on light emitted
from probe 2 upon incubation with the lowest concentrations of β-galactosidase.
Probes 2 and 3 ability to image β-galactosidase activity was initially evaluated in
aqueous solution (PBS, pH 7.4) using probe 1 as a control (Figure 10A). In the
absence of β-galactosidase, no chemiluminescence was observed from any of the
probes; however, in the presence of the enzyme, probes 2 and 3 emitted light with
much stronger intensity than that of probe 1 (about 100-fold for probe 2 and 25-fold
for probe 3, see Figure 10B). Probes 2 and 3 were selected for further evaluation in
vivo; notably, probe 3 which emits light within the near infrared (NIR) region. This
region of light is optimal for in vivo imaging applications since NIR photons penetrate
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organic tissues.38-43 Probes 2 and 3 were incubated with β-galactosidase and then
injected subcutaneously to mice. Under such conditions, clear chemiluminescence
images were obtained for both probes (Figure 10C), however the signal intensity
obtained from probe 3 was about 6-fold higher than that obtained from probe 2
(Figure 10D). No chemiluminescence signal was obtained from the probes without
pre-incubation with β-galactosidase.
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Figure 10. (A) Solution images obtained from 1 μM probes 1, 2 and 3 incubated in PBS, pH
7.4 in the presence and in the absence of β-galactosidase. (B) Quantification of signal
intensities obtained in solution in the presence of β-galactosidase. (C) Whole-body images
obtained 15 min following subcutaneous injection of probes 2 and 3 [50 µL, 1 µM in PBS
[100 mM], pH 7.4 after 30 min pre-incubation with or without 1.5 units/mL β-galactosidase].
(D) Quantification of signal intensities in whole-body images in the presence of β-
galactosidase (quantitative data are based on repeated imaging experiments with three
mice).
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In order to further compare the in vivo chemiluminescence signals of probes 2 and 3,
the probes (with and without ex vivo incubation with β-galactosidase) were injected
into mice via the intraperitoneal route. Remarkably, probe 3 produced a strong
chemiluminescence in vivo image, whereas no chemiluminescence signal was
observed for probe 2 (Figure 11). These observations clearly demonstrate the in vivo
imaging advantage of the NIR chemiluminescence produced by probe 3 vs. that of
the green wavelength produced by probe 2.
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Figure 11: Whole-body images obtained 15 min following intraperitoneal injection of probes
2 and 3 [50 µL, 1 µM in PBS [100 mM], pH 7.4 after a 30 min pre-incubation with or without
1.5 units/mL β-galactosidase].
In previous examples that used Schaap's dioxetanes for in vivo imaging,44-46
a
surfactant-dye adduct (Emerald-IITM enhancer) was added to the injected solution in
order to allow detection of the chemiluminescence signal. The use of multi-
component system for in vivo imaging has its obvious limitations, especially when
the animal is treated systemically. As we demonstrated in Figure 11, bright image
was obtained from the QCy-tethered dioxetane (probe 3), following ex-vivo
activation with β-galactosidase and intraperitoneal injection into mice. Here we
established the proof of concept of the dioxetane-fluorophore conjugates to act as
turn-ON chemiluminescent probes for in vivo imaging. In the next step, we intend to
study the capability of the probes to image real pathological events, such as cancer
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and inflammation. Yet, to demonstrate imaging based on real endogenous activity,
we next sought to image cells that endogenously overexpressed β-galactosidase.
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Cell-imaging using chemiluminescence microscopy by probe 3:
While fluorescence microscopy is a well-known established method for cell-imaging,
bioluminescence microscopy has recently been emerged.47 Instrumentation
improvements led to the development of the LV200 microscope by Olympus. The
setup of this microscope has significantly improved the ability to localize and
quantify luminescence probes at single cell resolution. So far, only luciferin was
demonstrated as a probe to image cells transfected by the luciferase gene. We
sought to evaluate the ability of probe 3 to image cells with overexpression of β-
galactosidase, by the LV200 microscope. HEK293 (transfected by LacZ) and HEK293-
WT (control) cells were incubated with probe 3 and then imaged by the LV200
(Figure 12) using a 20X objective (NA 0.75). Probe 3 was able to produce
chemiluminescence images of the HEK293 cells (Figure 12b), while no
chemiluminescence signal at all was observed by the HEK293-WT cells (Figure 12d).
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Figure 12: (a) Transmitted light image and (b) chemiluminescence microscopy of HEK293-
LacZ stable cells. (c) Transmitted light image and (d) chemiluminescence microscopy of
HEK293-WT cells. Images were obtained following 20 min incubation with cell culture
medium containing probe 3 (5 μM).
To improve the image quality, the HEK293-LacZ cells were fixed by using 4%
formaldehyde and permeabilized with 0.1% triton x-100. Then, the cells were
incubated with probe 3 and imaged by the microscope using a 60x objective (NA
1.42). As can be seen in Figure 13 ((a)-transmitted light, (b)-chemiluminescence), the
cells became visible, exhibiting a clear chemiluminescence emission.
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Figure 13: (a) Transmitted light image and (b) chemiluminescence microscopy of HEK293-
LacZ stable cells, fixed with formaldehyde (4% for 20 min). Images were obtained following
20 min incubation with cell culture medium containing probe 3 (5 μM).
Although the quality of the obtained images is not high yet, as far as we know, these
are the first chemiluminescence cell-images produced by a turn-ON small-molecule
probe that is not related to luciferin. This approach should allow to image other
enzymatic/chemical reactivities in cells by replacing the triggering group of the probe
with the appropriate analyte-responsive substrates. The synthetic strategy
developed in this work enables convenient synthesis of various chemiluminescence
probes. For example, a lipase or an esterase probe can be prepared by incorporation
of an appropriate ester triggering group instead of the β-galactose one. Similarly, the
synthesis of proteases probes can be achieved by using a short specific peptide as
triggering substrate. Of course incorporation of certain substrates could be more
challenging than others, however, the use of orthogonal protecting groups should
assist in solving synthetic difficulties.
Conclusions
In summary, we have developed a simple practical synthetic route for preparation of
turn-ON fluorophore-tethered dioxetane chemiluminescent probes. The
effectiveness of the synthesis is based on a late-stage functionalization of a
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dioxetane precursor by Hartwig-Miyaura C-H borylation, followed by subsequent
Suzuki coupling and oxidation to dioxetane. The obtained intermediate is composed
of a reactive NHS-ester-dioxetane ready for conjugation with any fluorophore-amine
derivative. We also reported the phenomenon of light-induced decomposition of
dioxetane-fluorophore conjugates, which highlights the advantage of our synthetic
method. The chemiluminescent emission of the fluorophore-tethered dioxetane
probes was significantly amplified in comparison to a classic dioxetane probe
through an energy transfer mechanism. The synthesized probes produced light of
various colors that matched the emission wavelength of the excited tethered
fluorophore. Using our synthetic route, we synthesized two fluorophore-tethered
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dioxetane probes designed for activation by β-galactosidase and conjugated with
green (fluorescein) and NIR (QCy) fluorescent dyes. Both probes were able to
provide chemiluminescence in vivo images following subcutaneous injection after
activation by β-galactosidase. However, chemiluminescence image following
intraperitoneal injection was observed only by the NIR probe. These are the first in
vivo images produced by Schaap's dioxetane-based chemiluminescence probes with
no need of any additive. The NIR probe was also able to image cells, by
chemiluminescence microscopy, based on endogenous activity of β-galactosidase.
Such probes could be used for in vivo imaging of reporter genes, enzymes and
chemical analytes. We anticipate that our practical synthetic methodology for
dioxetane-tethered building blocks will be useful for preparation of various
chemiluminescent probes suitable for numerous applications.
Supporting Information Available: Synthetic procedures, characterization data of all
new compounds, chemiluminescence spectrum of probe 1; NMR and MS spectra of
probes 1-3; Experimental details of in vivo evaluations and cell microscopy imaging
(PDF). This material is available free of charge via the internet at http://pubs.acs.org.
Acknowledgements: D.S. thanks the Israel Science Foundation (ISF), the Binational
Science Foundation (BSF) and the German Israeli Foundation (GIF) for financial
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support. This work is supported in part by a grant from the Israeli National
Nanotechnology Initiative in the Focal Technology Area: Nanomedicines for
Personalized Theranostics. R.S.F. thanks the European Research Council for the ERC
Consolidator Grant Agreement n. [617445]- PolyDorm.
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References
(1)
(2)
Roda, A.; Guardigli, M. Anal. Bioanal. Chem. 2012, 402, 69.
Roda, A.; Guardigli, M.; Pasini, P.; Mirasoli, M.; Michelini, E.; Musiani,
M. Anal. Chim. Acta 2005, 541, 25.
(3)
Gross, S.; Gammon, S. T.; Moss, B. L.; Rauch, D.; Harding, J.; Heinecke,
J. W.; Ratner, L.; Piwnica-Worms, D. Nat. Med. 2009, 15, 455.
(4)
Zhang, N.; Francis, K. P.; Prakash, A.; Ansaldi, D. Nat. Med. 2013, 19,
500.
(5)
Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. J. Am. Chem. Soc.
2013, 135, 1783.
(6)
Porterfield, W. B.; Jones, K. A.; McCutcheon, D. C.; Prescher, J. A. J.
Am. Chem. Soc. 2015, 137, 8656.
(7)
Merényi, G.; Lind, J.; Eriksen, T. E. J. Biolumin. Chemilumin. 1990, 5,
53.
(8)
Silva, S. M.; Wagner, K.; Weiss, D.; Beckert, R.; Stevani, C. V.; Baader,
W. J. Luminescence 2002, 17, 362.
(9)
Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros,
J.; Taylor, W. R.; Murthy, N. Nat. Mater. 2007, 6, 765.
(10) Kielland, A.; Blom, T.; Nandakumar, K. S.; Holmdahl, R.; Blomhoff, R.;
Carlsen, H. Free Radical Biol. Med. 2009, 47, 760.
(11) Lim, C.-K.; Lee, Y.-D.; Na, J.; Oh, J. M.; Her, S.; Kim, K.; Choi, K.; Kim, S.;
Kwon, I. C. Adv. Funct. Mater. 2010, 20, 2644.
(12) Cho, S.; Hwang, O.; Lee, I.; Lee, G.; Yoo, D.; Khang, G.; Kang, P. M.; Lee,
D. Adv. Funct. Mater. 2012, 22, 4038.
(13) Lee, Y.-D.; Lim, C.-K.; Singh, A.; Koh, J.; Kim, J.; Kwon, I. C.; Kim, S. ACS
nano 2012, 6, 6759.
(14) Shuhendler, A. J.; Pu, K.; Cui, L.; Uetrecht, J. P.; Rao, J. Nat. Biotechnol.
2014, 32, 373.
(15) Lee, E. S.; Deepagan, V. G.; You, D. G.; Jeon, J.; Yi, G. R.; Lee, J. Y.; Lee,
D. S.; Suh, Y. D.; Park, J. H. Chem. Commun. 2016, 52, 4132.
(16) Li, P.; Liu, L.; Xiao, H.; Zhang, W.; Wang, L.; Tang, B. J. Am. Chem. Soc.
2016, 138, 2893.
(17) Schaap, A. P.; Handley, R. S.; Giri, B. P. Tetrahedron Lett. 1987, 28,
935.
(18) Schaap, A. P.; Chen, T.-S.; Handley, R. S.; DeSilva, R.; Giri, B. P.
Tetrahedron Lett. 1987, 28, 1155.
20
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Journal of the American Chemical Society
1
2
3
4
5
6
7
8
(19) Schaap, A. P.; Sandison, M. D.; Handley, R. S. Tetrahedron Lett. 1987,
28, 1159.
(20) Matsumoto, M. J. Photochem. Photobiol., C 2004, 5, 27.
(21) Park, J. Y.; Gunpat, J.; Liu, L.; Edwards, B.; Christie, A.; Xie, X. J.; Kricka,
L. J.; Mason, R. P. Luminescence 2014, 29, 553.
(22) Tseng, J. C.; Kung, A. L. J. Biomed. Sci. 2015, 22, 45.
(23) Schaap, A. P.; Akhavan, H.; Romano, L. J. Clin. Chem. 1989, 35, 1863.
(24) Dominguez, A.; Fernandez, A.; Gonzalez, N.; Iglesias, E.; Montenegro,
L. J. Chem. Educ. 1997, 74, 1227.
(25) Torchilin, V. P. J. Controlled Release 2001, 73, 137.
(26) Schaap, A. P.; Akhavan-Tafti, H. patent WO 1990007511 A1 1990.
(27) Watanabe, N.; Kino, H.; Watanabe, S.; Ijuin, H. K.; Yamada, M.;
Matsumoto, M. Tetrahedron 2012, 68, 6079.
(28) Matsumoto, M.; Watanabe, N.; Hoshiya, N.; Ijuin, H. K. Chem. Rec.
2008, 8, 213.
(29) Loening, A. M.; Dragulescu-Andrasi, A.; Gambhir, S. S. Nat. Methods
2010, 7, 5.
(30) Branchini, B. R.; Ablamsky, D. M.; Rosenberg, J. C. Bioconjugate Chem.
2010, 21, 2023.
(31) McCutcheon, D. C.; Paley, M. A.; Steinhardt, R. C.; Prescher, J. A. J.
Am. Chem. Soc. 2012, 134, 7604.
(32) Jathoul, A. P.; Grounds, H.; Anderson, J. C.; Pule, M. A. Angew. Chem.,
Int. Ed. 2014, 53, 13059.
(33) Steinhardt, R. C.; O'Neill, J. M.; Rathbun, C. M.; McCutcheon, D. C.;
Paley, M. A.; Prescher , J. A. Chem. - Eur. J. 2016, 22, 3671.
(34) Ishiyama, T.; Takagi, J.; Ishida, K.; Miyaura, N.; Anastasi, N. R.; Hartwig,
J. F. J. Am. Chem. Soc. 2002, 124, 390.
(35) Karton-Lifshin, N.; Segal, E.; Omer, L.; Portnoy, M.; Satchi-Fainaro, R.;
Shabat, D. J. Am. Chem. Soc. 2011, 133, 10960.
(36) Karton-Lifshin, N.; Albertazzi, L.; Bendikov, M.; Baran, P. S.; Shabat, D.
J. Am. Chem. Soc. 2012, 134, 20412.
(37) Edwards, B.; Sparks, A.; Voyta, J. C.; Bronstein, I. in Bioluminescence
and chemiluminescence: fundamentals and applied aspects (A. K. Campbell, L. J.
kricka and P. E. Stanley, eds.); Wiley: Chichester, 1994, p. 56.
(38) Weissleder, R. Nat. Biotechnol. 2001, 19, 316.
(39) Gnaim, S.; Shabat, D. Acc. Chem. Res. 2014, 47, 2970.
(40) Redy-Keisar, O.; Kisin-Finfer, E.; Ferber, S.; Satchi-Fainaro, R.; Shabat,
D. Nat. Protocols 2014, 9, 27.
(41) Kisin-Finfer, E.; Ferber, S.; Blau, R.; Satchi-Fainaro, R.; Shabat, D.
Bioorg. Med. Chem. Lett. 2014, 24, 2453.
(42) Redy-Keisar, O.; Huth, K.; Vogel, U.; Lepenies, B.; Seeberger, P. H.;
Haag, R.; Shabat, D. Org. Biomol. Chem. 2015, 13, 4727.
(43) Redy-Keisar, O.; Ferber, S.; Satchi-Fainaro, R.; Shabat, D.
ChemMedChem 2015, 10, 999.
9
10
11
12
13
14
15
16
17
18
19
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(44) Cao, J.; Campbell, J.; Liu, L.; Mason, R. P.; Lippert, A. R. Anal. Chem.
2016, 88, 4995.
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(45) Cao, J.; Lopez, R.; Thacker, J. M.; Moon, J. Y.; Jiang, C.; Morris, S. N.;
Bauer, J. H.; Tao, P.; Mason, R. P.; Lippert, A. R. Chem. Sci. 2015, 6, 1979.
(46) Liu, L.; Mason, R. P. PLoS One 2010, 5, e12024.
(47) Bauer, C. R. G. I. T. Imaging & Microscopy 2013, 4, 32.
(48) Wakimoto, R.; Kitamura, T.; Ito, F.; Usami, H.; Moriwaki, H. Appl.
Catal., B 2015, 166–167, 544.
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