Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation

Article abstract of DOI:10.1016/j.cclet.2016.02.011

Replacement of the methylene group at the C-8 position with an extended electronic conjugation is a new promising method to develop red-shifted coelenterazine derivatives. In this paper, we have described an oxygen-containing coelenterazine derivative with a significant red-shifted (63 nm) bioluminescence signal maximum relative to coelenterazine 400a (DeepBlueC, 1). In cell imaging, the sulfur-containing coelenterazine derivative displayed a significantly (1.77 ± 0.09; P ≤ 0.01) higher luminescence signal compared to coelenterazine 400a and the oxygen-containing coelenterazine derivative exhibited a slightly (0.74 ± 0.08; P ≤ 0.05) lower luminescence signal. It is beneficial to understand further the underlying mechanisms of bioluminescence.

Full text of DOI:10.1016/j.cclet.2016.02.011

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Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Luminescence of coelenterazine derivatives with C-8 extended
electronic conjugation
*
Ming-Liang Yuan, Tian-Yu Jiang, Lu-Pei Du, Min-Yong Li
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Replacement of the methylene group at the C-8 position with an extended electronic conjugation is a
new promising method to develop red-shifted coelenterazine derivatives. In this paper, we have
described an oxygen-containing coelenterazine derivative with a significant red-shifted (63 nm)
bioluminescence signal maximum relative to coelenterazine 400a (DeepBlueCTM, 1). In cell imaging, the
sulfur-containing coelenterazine derivative displayed a significantly (1.77 Æ 0.09; P ꢁ 0.01) higher
luminescence signal compared to coelenterazine 400a and the oxygen-containing coelenterazine derivative
exhibited a slightly (0.74 Æ 0.08; P ꢁ 0.05) lower luminescence signal. It is beneficial to understand further
the underlying mechanisms of bioluminescence.
Received 14 January 2016
Received in revised form 18 February 2016
Accepted 19 February 2016
Available online xxx
Keywords:
Bioluminescence imaging
Coelenterazine derivatives
Relative quantum yield
Chemiluminescence
Red-shifted
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
(450–475 nm). To overcome such a limitation, much effort has
been made either by modifications of the RLuc [9] or by synthesis
Noninvasive bioluminescence imaging (BLI) has emerged as a
routine technique in fields such as medical science, biochemistry
and molecular biology [1–3]. Wide BLI applications have been
discovered including real-time monitoring of gene expression [4],
tumor growth [5] and protein–protein interactions [6]. The
mechanism depends on an enzymatic oxidation reaction involving
an enzyme (luciferase), a substrate (luciferin) and molecular
oxygen, resulting in the emission of light.
of new red-shifted CTZ analogues [10]. To our knowledge, it seems
to be difficult to achieve red-shifted variants of RLuc via molecular
biology approaches. Hence, we focus on the modifications of CTZ
analogues to find a bathochromic and stable substrate of Rluc.
Since the enzymatic recognition mechanism of the Rluc system is
not yet fully understood [11], the design of novel valuable CTZ
derivatives is challenging, and only few red-shifted substrates have
been reported so far. One significant molecule, v-coelenterazine
To avoid absorption and scattering by tissue in small-animal
imaging experiments, the wavelength range of light emissions had
better be the red to near-infrared (600–900 nm). Firefly luciferase
(Fluc, 61 kDa) is the most commonly utilized BLI system due to its
favorable emission spectrum (emission spectra in the range of
540–615 nm), stability, and biocompatibility of its bioluminogenic
owning a more planar and rigid molecular structure with a vinylene
bridge at the C-5 position, displays a remarkable red-shifted
emission at 512 nm and high quantum yield with RLuc, however
at the cost of synthesis and molecular stability [10]. As to other CTZ
derivatives modified at the C-2 [12], C-5 [10], C-6 [13] and C-8 [14]
positions of the imidazopyrazinone core, few show luminescence
properties superior to those of native CTZ, mostly since their
structural modifications prevent their enzymatic recognition. In
previous work, the luminescence properties of many modified CTZ
derivatives are measured in comparison with coelenterazine 400a
(DeepBlueCTM, 1). Coelenterazine 400a, a commercially available
CTZ derivative, produces a ꢀ400 nm emission peak upon Rluc-
mediated oxidation. Itisused for bioluminescenceresonanceenergy
transfer (BRET) studies because it has minimal interference with the
emission of the GFP acceptor, which provides greater signal
resolution [6]. Recently, a sulfur-containing CTZ derivative 3
(Fig. 1) was discovered with a 30 nm red-shift compared to
coelenterazine 400a in bioluminescence [15]. At the same time,
substrate D-luciferin [7]. Unfortunately, however, cofactors such as
ATP and Mg2+ ion are required which potentially leading to the
experimental complexity in bioanalysis. On the other hand, renilla
luciferases (Rluc, 36 kDa) in conjunction with coelenterazine (CTZ)
or its derivatives as the substrate only require molecular oxygen
[8]. Moreover, Rluc has been successfully expressed in mammalian
cells without any cytotoxicity. However, the Rluc/CTZ pair
produces a blue–green light with are latively short wavelength
*
Corresponding author.
E-mail address: mli@sdu.edu.cn (M.-Y. Li).
http://dx.doi.org/10.1016/j.cclet.2016.02.011
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: M.-L. Yuan, et al., Luminescence of coelenterazine derivatives with C-8 extended electronic
conjugation, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.011

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the relative quantum yield (RQY) is 3.6 compared to coelenterazine
400a, measured by mixing substrates and rough cytosolic extract
prepared from cells expressing Rluc. However, in cells there are
many unwanted substances prone to combine with CTZ derivatives
and catalyze the oxidation reaction such as albumin and other
intrinsic proteins besides Rluc, thus giving rise to background noise
in bioluminescence assays [16].
In this paper, we designed and synthesized a CTZ derivative that
contains an oxygen atom in place of the native methylene group at
C-8 position (1). We hypothesized that this replacement would
also produce a red-shifted emission because of similar attributes of
the oxygen and sulfuratom. To examine RQY, a new measurement
method through an IVIS Kinetic equipped with a cooled charge-
coupled-device (CCD) detector had been developed. Besides we
employed commercially available purified Rluc enzyme in place of
the rough cytosolic extract to decrease enzyme-independent
luminescence (autoluminescence) of CTZ derivatives. To further
evaluate the luminescence properties of CTZ derivatives, we
performed the assay for luminescence activity in the live cell. All
results showed that CTZ derivative 2 (Fig. 1) displayed a more
significant red-shift (63 nm) in bioluminescence compared to
coelenterazine 400a while it had lower quantum yield. In cell
camera. All the experiments were carried out at room temperature
unless otherwise specified.
2.2. Synthesis of CTZ derivatives 1–3
The synthesis of compound 1–3 was shown in Schemes S1 and
S2 in Supporting information. The details for preparation and NMR
and HRMS spectra of these compounds were also presented in
Supporting information.
2.3. Luminescence spectral analysis
Synthesized CTZ derivatives were dissolved in ethanol at a
concentration of 1 mg/mL foruse as stock solutions, stored at À20 8C
or lower temperature (for long-time storage), and diluted in
50 mmol/L Tris–HCl buffer pH 7.4 (without calcium and magne-
sium) immediately prior to use. Enzyme assays were conducted
using commercially available purified Rluc enzyme (Ray Biotech) at
a concentration of 28 nmol/L in 1 mL PBS (pH 7.4) stored at À80 8C.
Luminescence spectra were recorded using an F-2500 FL
Spectrophotometer with the excitation lamp turned off and the
emission shutter open at a scanning speed of 3000 nm/min. All the
spectra were measured at room temperature. The in vitro
bioluminescence spectra were measured in 50 mmol/L Tris–HCl
buffer pH 7.4 containing Rluc protein at a final concentration of
15 nmol/L and initiated by the injection of CTZ derivatives buffer
imaging photon emission from
3 was significantly higher
compared to coelenterazine 400a (1.77 Æ 0.09; P ꢁ 0.01), while
compound 2 exhibited a 0.74 Æ 0.08 slightly lower luminescence
signal. Therefore, replacement of the methylene group at the C-8
position with an O or S heteroatom is a new promising method to
develop red-shifted CTZ analogs.
solution at a final concentration of 50
spectra were traced by mixing 200
solution at a final concentration of 50
m
m
mol/L. Chemiluminescence
L of CTZ derivatives buffer
mol/L with 800 L of
m
m
dimethylsulfoxide (DMSO). The response time is 2.0 s, and all
spectra were not corrected for luminescence decay at spectral
scanning. The luminescence spectra are the result of three
independent measurements, each one measured in triplicate.
2. Experimental
2.1. Materials and apparatus
All reagents for organic synthesis were obtained from
commercial suppliers and used without further purification. When
necessary, organic solvents were routinely dried and/or distilled
prior to use and stored over molecular sieves under argon.
Millipore water was used to prepare all aqueous solutions. 1H NMR
and 13C NMR were recorded on Bruker AV-300 or AV-600
spectrometers at the College of Chemistry NMR Facility, Shandong
2.4. Relative quantum yield (RQY) and kinetics of in vitro
luminescence
The RQY study and kinetic analysis were performed using an
IVIS Kinetic (Caliper Life Sciences, USA) which consisted of a cooled
charge-coupled device (CCD) camera mounted on a light-tight
specimen chamber (dark box), a camera controller, a camera
cooling system, and controlled using a computer. The data are
represented as pseudocolor images (in photons/s/cm2/scr) of light
intensity (blue—least intense, red—most intense) superimposed
over the grayscale reference images. Circular specified regions of
interest (ROIs) were drawnon the areas, and the light output were
quantified as the total number of photons emitted per second using
Living Image software.
University. All chemical shifts are reported in the standard
d
notation of parts per million using the peaks of residual proton and
carbon signals of the solvent as internal references. Mass spectra
were recorded in ESI+ mode (70 eV) in Drug Analysis Center at
Shandong University. ESI-HRMS was performed on a Waters
SYNAPT G2-Si. The purity of CTZ analogues was confirmed by
analytical reverse-phased HPLC (Agilent, 1260 Infinity) on
Phenomenex C-18 column (250 Â 4.6 mm). Melting points were
determined on a Mel-Temp apparatus and were not corrected.
Luminescence spectra were recorded using an F-2500 FL Spectro-
photometer. The light outputs were determined with an IVIS
Kinetic (Caliper Life Sciences, USA) equipped with a cooled CCD
To determine the appropriate unsaturated amount of substrate,
10
mL of CTZ derivatives 1–3 between 1 and 100 mmol/L and 90 mL
of either DMSO (chemiluminescence) or Rluc enzyme at a final
concentration of 15 nmol/L (bioluminescence) were used. The RQY
and reaction kinetics was determined by mixing 10
m
L of CTZ
derivatives (final concentration of 5 mol/L) with 90
m
mL of either
DMSO or Rluc enzyme (15 nmol/L) onto wells of 96-well black
plates to prevent light reflection from well to well. Luminescent
signals were measured immediately after mixing and monitored
over a period of 25–30 min (luminescence had almost decayed to
near-background levels) using the IVIS. Light output was recorded
every 5 min with an exposure time of 30 s for chemiluminescence
and every 1 min with an exposuretime of 5 s in the first 15 min for
bioluminescence. The collected data was analyzed by employing
the Prism 5.0 GraphPad software to compute the total light output.
As a corresponding blank control, Tris–HCl buffer was added
instead of CTZ derivatives solution under the same conditions. All
assays were performed in triplicate.
Fig. 1. Structures of the coelenterazine analogs 1–3.
Please cite this article in press as: M.-L. Yuan, et al., Luminescence of coelenterazine derivatives with C-8 extended electronic
conjugation, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.011

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3
2.5. Assay for luminescence activity in live cell
real experiments, QY is achieved by dividing the absolute number
of all emitted photons by the number of substrate molecules
consumed. However, because of different system manufacture and
measuring conditions, it is difficult to gain a standard value of QY
and it need not to for the purpose of this paper. Hence, we
evaluated the luminescence properties by another related
parameter, the relative quantum yield (RQY).
ES-2 cells (human ovarian cancers cell line) expressing Rluc
were supplied by BioDiagnosis. The ES-2 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; high glucose with
L-glutamine; Gibco) containing 10% fetal bovine serum (FBS) and
0.5 g/mL puromycin at 37 8C in a humidified atmosphere in a 5%
m
CO2 incubator. CTZ derivatives was dissolved in ethanol to make a
1 mmol/L stock solution, and diluted with Tris–HCl buffer to
In this paper, we developed a new measurement method
through an IVIS Kinetic equipped with a cooled charge-coupled-
device (CCD) detector as a sensitive photodetector to examine QY
or RQY. The CCD detector has prominent advantages over
traditional photomultiplier tubes (PMTs) [18], such as high
quantum efficiency, low direct current noises, and parallel
multichannel photo-detection, which are very appropriate for
measuring efficiently.
gradient concentration (5, 10, 20, 40, 60, 80, 100
mmol/L).
The ES-2-Rluc cells were grown in black 96-well plates (4 Â 104
cells per well). After a 24-h incubation period, the medium was
removed. Then cells were washed with Tris–HCl buffer twice and
treated with 100
solutions (ranging from 0 to 100
m
L of a series of concentrations of CTZ derivatives
mol/L). Bioluminescent signals
m
were then immediately determined using the IVIS. Light output
was recorded every 1 min with an exposure time of 30 s until
luminescence almost decayed to near-background levels. Lumi-
nescent signal (photons per second) for each well was measured
and plotted as average values. All experiments were performed in
triplicate.
Before acquiring RQY, we performed the kinetic analysis of in
vitro luminescence reactions and plotted kinetic profile of bio/
chemiluminescence presented as Fig. 3 and Fig. S1 in Supporting
information. In coordinates, the vertical axis expresses the
absolute number of photons per second, and the horizontal axis
shows the reaction time, thus the area under the curve (AUC)
meaning the absolute total number of luminescence photons. The
absolute total number of photons divided by the number of reacted
molecules gives QY. As a result, we also obtain RQY (relative to
coelenterazine 400a).
3. Results and discussion
3.1. Luminescence spectra
To determine the appropriate amount of substrate and discover
anoptimized conditionto measureRQY, we setup the concentration
The luminescence spectra of CTZ derivatives 1, 2 and 3 were
determined using dimethylsulfoxide (DMSO) for chemilumines-
cence and Rluc enzyme for bioluminescence. As shown in Fig. 2a
and Table S1 in Supporting information, sulfur-containing CTZ
derivative 3 produced a 42 nm red-shifted spectrum measured in
neutral DMSO relative to coelenterazine 400a (compound 1).
However, oxygen-containing CTZ derivative 2 displayed a 10 nm
blue-shift in chemiluminescence. Significantly, the sulfur and
oxygen heteroatom replacement of the methylene bridge in
coelenterazine 400a both lead to a bathochromic shift in the
bioluminescent reaction with Rluc. The bioluminescence spectra
exhibited a 36 nm red-shift for 3 and more significant red-shift
(63 nm) for 2 compared to coelenterazine 400a (Fig. 2b). As a
result, the differences between the emission peaks were generally
of CTZ derivatives 1–3 ranging from 1
mmol/L to 100 mmol/L. As a
result, the concentration at 5 mol/L is suitable for both chemilu-
m
minescence and bioluminescence according to Fig. 3. First, RQY of
CTZ derivatives 1–3 for chemiluminescence were measured, and it
was 1.81 to compound 3, which is in good agreement with
previously reported value [15]. While RQY was 0.77 to compound 2
inferior to coelenterazine 400a, the luminescence intensity of 2 was
higher than coelenterazine 400a during the first 5 min. In the
bioluminescence reaction, compounds 2 and 3 showed lower
quantum yield (Table S1). Nevertheless, compound 3 owned a
relative lower saturated concentration, indicating that it had a
higher affinity to native renilla luciferase (Rluc). Meanwhile, 2 and 3
may show better bioluminescence intensities using Rluc variants
such as the widely known Rluc 8 and Rluc 8.6 variants [9,19] than
coelenterazine 400a, which needed further study. Therefore, 2 and 3
are promising bright red-shifted CTZ derivatives.
attributed to an effective extension of
p-electron conjugation,
electronegativity, or H-bonds within the active site of oxygen and
sulfur heteroatom at the C-8 position [17].
Measurements of the chemiluminescence and bioluminescence
emission time profiles revealed that all three CTZ derivatives show
the typical flash-type luminescence kinetics [20]. For 1, an
immediate burst to peak emission rate followed by steady decay
and for 2 and 3, a same immediate burst to peak emission rate but
followed by a relatively dramatic decay with shorter half-life times
in both chemiluminescence and bioluminescence reactions, likely
3.2. Relative quantum yield (RQY) and kinetics of in vitro
luminescence
The quantum yield (QY) in bio/chemiluminescence is defined as
the efficiency of the production of a photon from a single reactant
molecule and is a key quantity to characterize the reaction [18]. In
Fig. 2. (a) Chemiluminescence emission spectra of coelenterazine derivatives; (b) Bioluminescence emission spectra of coelenterazine derivatives.
Please cite this article in press as: M.-L. Yuan, et al., Luminescence of coelenterazine derivatives with C-8 extended electronic
conjugation, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.011

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Fig. 3. Comparison of the chemiluminescence generated by the three CTZ derivatives in DMSO. (a) Dose-responsiveness of chemiluminescenceimmediately measured after
the addition of CTZ derivatives ranging from 1 mol/L to 100 mol/L using the IVIS; (b) Representative chemiluminescence images shown after the addition of CTZ
m
m
derivatives in 96-well plates; (c) Time course of light output following addition of 5
m
mol/L substrate using the IVIS; (d) The reaction kinetics of different CTZ derivatives in
chemiluminescence. All values are shown as mean Æ SD (n = 3).
due to an intrinsic change in the electronic properties of the excited
states of the molecule, or the different binding affinity of CTZ to Rluc.
Interestingly, photon emission from 3 was significantly higher
compared with 1 (1.77 Æ 0.09; P ꢁ 0.01). At the same time, 2 exhibited
a 0.74 Æ 0.08 lower luminescence signal compared with 1 (Fig. 4).
Considering low quantum yield in the bioluminescence reaction and
highquantumyieldinthechemiluminescencereaction forcompound 3,
we think 3 or other hydrophobic CTZ derivatives may produce enzyme-
independent luminescence (autoluminescence) in complex intracellu-
lar environment after permeating through the cell membrane, for
3.3. Luminescence activity in live cell
Next, we performed the assay for luminescence activity in live
ES-2 cells expressing Rluc. The total photons of the first 10 min
(i.e. AUC) were used to measuring luminescence signal intensity.
Fig. 4. Comparison of the luminescence generated by the three CTZ derivatives in ES-2 cells expressing Rluc. (a) Dose-responsiveness of luminescence immediately measured
after the addition of CTZ derivatives ranging from 5 to 100 mol/L; (b) Representative luminescence images shown after the addition of CTZ derivatives in 96-well plates; (c)
Time course of light output following addition of 40
m
m
mol/L substrate; (d) Photon flux comparison, compound 2 (* P ꢁ 0.05)showing the significantly lower photon emission
compared with coelenterazine 400a and compound 3 (** P ꢁ 0.01) showing the significantly higher photon emission compared with coelenterazine 400a. All values are
shown as mean Æ SD (n = 3).
Please cite this article in press as: M.-L. Yuan, et al., Luminescence of coelenterazine derivatives with C-8 extended electronic
conjugation, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.011

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example albumin can catalyze CTZ chemiluminescence consistent with
a monooxygenase [16]. Thus, a higher luminescence signal was
recorded compared with a pure bioluminescence.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.02.
011.
Please cite this article in press as: M.-L. Yuan, et al., Luminescence of coelenterazine derivatives with C-8 extended electronic
conjugation, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.02.011