An allylated firefly luciferin analogue with luciferase specific response in living cells

Article abstract of DOI:10.1039/c7cc09720d

An allylated firefly luciferin was successfully synthesized and its bioluminescence properties were evaluated. When applied to cellular imaging in combination with Eluc, which is one of the commercially available luciferases, this analogue displayed a luciferase-specific bioluminescence signal with prolonged emission (>100 min).

Full text of DOI:10.1039/c7cc09720d

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K. Niwa, T. Nakajima, N. Kitada, S. A. Maki, M. Sato, D. Citterio, S. Nishiyama and K. Suzuki, Chem.
Commun., 2018, DOI: 10.1039/C7CC09720D.
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An Allylated Firefly Luciferin Analogue with Luciferase Specific
Response in Living Cells
Yuma Ikeda,a Tsuyoshi Saitoh,b Kazuki Niwa,c Takahiro Nakajima, d Nobuo Kitada, e Shojiro A.
Maki,e Moritoshi Sato,d Daniel Citterio,*a Shigeru Nishiyamaf and Koji Suzuki*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
An allylated firefly luciferin was succesfully synthesized and its
bioluminescence properties were evaluated. When applied to
cellular imaging in combination with Eluc, which is one of the
commercially available luciferases, this analogue displayed a
luciferase-specific bioluminescence signal with prolonged
emission (>100 min).
a)
Luciferase
ATP, Mg2+
PPi
+
D-luciferin
D-luciferyl-AMP
O2
AMP CO
+
hν
+
+
2
D-luciferyl-AMP
Oxyluciferin
Bioluminescence-based assays with luciferin-luciferase pairs
are widely used as in vitro and in vivo reporters of biologically
relevant substances, gene expression, and tumour
progression, among others.1,2 Since bioluminescence-based
assays do not require excitation light, they exhibit much lower
background signals than fluorescence-based assays and are
applicable to highly sensitive bioimaging.3 Among the available
systems, the firefly luciferin-luciferase reaction is one of the
most well-studied emission systems, due to its high quantum
yield (φBL = 0.23~0.61) and relatively long emission wavelength
(λem 530~620 nm).4 In the firefly bioluminescence reaction, the
b)
7`
D-luciferin
7`-AllylLuc
Fig. 1 a) Firefly bioluminescence reaction: Luciferase catalyses the adenylation and
oxidation of its substrate D-luciferin to release a photon. b) Chemical structures of D-
luciferin and 7`-AllylLuc.
animals utilizing their different bioluminescence colours and
superior properties, such as prolonged light emission and high
luminescence efficiency, among others.8,9 However, it is widely
luciferase catalyses the oxidation of D-luciferin in the presence
of adenosine triphosphate (ATP) and Mg2+ acting as co-factors
to release a light photon. (Fig. 1a).
known that D-luciferin is the only substrate that results in light
A variety of luciferases has been discovered5–7 and cloned,
and many of them have been adapted to “spy” on cells in
whole
emission in combination with these luciferases. Although
different luciferases can be selectively expressed to a specific
target, they all result in bioluminescence emission upon
introduction of D-luciferin. For this reason, it is basically not
possible to distinguish between multiple targets within a single
cell. Although the development of luciferases has been a very
active field of research, there are unfortunately only few
reports of successful development of synthetic luciferins,
presumably due to the difficulty in designing and synthesizing
bioluminescence-active luciferins.
Many details regarding bioluminescence of the firefly system
still remain unknown, and random modifications of the
luciferin structure may lead to the loss of the luminescence
activity. Therefore, we referred to previously performed
studies on the structure-activity relationship of firefly luciferin
to more efficiently design analogues. The molecular structure
of
D-luciferin is mainly composed of two subunits, the
thiazoline and
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pyrꢀHCl
K2CO3
neat, 200 ℃
DMF, RT
1
2, 77%
3, 96%
Δ
D-Cys
MeOH/H2O (pH 8.0)
RT
180 ℃
4, 95% (b.r.s.m)
7`-AllylLuc, 97%
Scheme1 Synthesis of 7`-AllylLuc
the benzothiazole substructures. The position and the
stereochemistry of the carboxyl group on the thiazoline ring is
a crucial factor for the luminescence activity.10–12 In contrast to
the luminescence activity of the
D-form, it is widely known
that the optical isomer - luciferin works as a potential
L
luminescence inhibitor.13 Additionally, many of the
heteroatom-variants and substituent modifications of this
thiazoline ring have resulted in a dramatic reduction or loss of
luminescence activity,14,15 and thus, the thiazoline ring subunit
is regarded as an essential site for substrate recognition by the
luciferase enzyme and the luminescence activity. On the other
hand, it has been reported that most analogues with
modification at the benzothiazole ring subunit retain their
luminescent activities. Actually, analogues with heteroatom-
substitution in the benzothiazole ring15,16 and modification of
To explore the potential emission ability of 7`-AllylLuc, a
conventional chemiluminescence assay was first conducted.
This non-enzymatic emission process is a useful
Fig. 2 Normalized bioluminescence emission spectra and light emission time courses
obtained with various luciferases for a) D-luciferin and b) 7`-AllylLuc at pH 8.0.
Additional experimental details are described in the ESI.
method for verifying the luminescence ability of the substrate
itself. When 7`-AllylLuc was subjected to chemiluminescence
its chemical structure17-19 maintained the luminescence assay conditions, luminescence was observed (Fig. S1). This
suggested that 7`-AllylLuc is a potential emitter for luciferases.
Next, it was investigated, whether firefly or beetle luciferases
are able to recognize 7`-AllylLuc as their substrate to release a
photon of light. Bioluminescence measurements with
commonly used luciferases were carried out using
commercially available enzymes, including firefly luciferase
(Fluc) from Photinus pyralis, Emerald Luc (Eluc) from
Pyrearinus termitilluminans, Stable Luciferase Green (SLG)
from Rhagophthalmus ohbai, as well as Stable Luciferase
Orange (SLO) and Stable Luciferase Red (SLR) from Phrixothrix
hirtus. 7`-AllylLuc exhibited bioluminescence catalysed by all of
these luciferases similar to that of the native luminescent
activity with shifts in the emission wavelength. Therefore, it
can be said that the benzothiazole ring is the modifiable site of
D-luciferin. A recent study revealed that modifications of the
benzothiazole ring at the C-7` position with sterically
demanding residues potentially lead to bolstered
bioluminescence properties.20 Furthermore, it was also shown
that mutant luciferases can effectively result in substrate-
specific luminescent properties with structurally modified
luciferin analogues.21,22
In order to expand the applications of bioluminescent
systems, we have developed a firefly luciferin analogue
modified with an allyl group at the C-7` position of its
benzothiazole core (Fig. 1b), exhibiting bioluminescence
substrate
the bioluminescence spectra of 7`-AllylLuc showed
significant red-shift (maximum emission wavelength at 605
nm) compared with that of -luciferin (Fig. 2 and S2), despite
D-luciferin (Fig. 2). Upon using Fluc as the luciferase,
a
emission with various luciferases in analogy to
D-luciferin, and
confirmed its utility for bioluminescence imaging.
D
having almost identical fluorescence spectra (Fig. S3), whereas
such a shift was not observed when using the other luciferases
(Eluc, SLG, SLO and SLR). Moreover, the bioluminescence
spectra obtained under several pH conditions with Fluc, known
to be a pH sensitive luciferase, were completely identical like
previously reported C-7` modified analogues (Fig. S4).17
In the crystalline structure of Luciola cruciate luciferase,
which is a type of luciferase with an amino acid sequence
resembling that of Fluc, complexed with an inhibitor (5`-O-[N-
(dehydroluciferyl)-sulfamoyl]adenosine; DLSA) structurally
To access the desired allyl-modified luciferin structure, focus
was set on the Claisen rearrangement for introduction of an
allyl-group (Scheme 1). The synthesis of allyl luciferin begins
with pyridine hydrochloride promoted thermal deprotection of
commercially available 2-cyano-6-methoxybenzothiazole (
which is transformed to its phenol form, 2-cyano-6-
hydroxybenzothiazole ( ). It is then converted to the allyloxy-
benzothiazole ( ) by treatment with allyl bromide/K2CO3,
which on regioselective Claisen rearrangement of the allyl
group results in compound ( ). The coupling reaction of 2-
cyano-benzothiazole derivative ( ) with -cysteine generated
1),
2
3
4
similar to D-luciferyl-AMP, it has been clarified that luciferin is
4
D
tightly sandwiched in the extremely hydrophobic
microenvironment of the enzyme.23 However, collapse of the
planar structure of luciferin by structural modification causes a
7`-AllylLuc via the standard procedure. Thus, 7`-AllylLuc can be
prepared in 4 steps from commercially available starting
materials in 68% overall yield.
disruption of the hydrogen-bonding network around the
17
luciferin,
which may result in
a
more polar
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microenvironment (Fig. S5). Finally, the energy level of the
excited state oxyluciferin is stabilized, and red-shifted
bioluminescence emission is observed. Actually, previous
studies have described
Encouraged by these results in vitro, the bioluminescent
properties of 7`-AllylLuc were further evaluated in live
luciferase-expressing COS-7 monkey kidney tissue cells. The
cells were cultured in 96-well microplates and monitored with
a luminometer over 100 minutes with various substrate
Table 1 Kinetic properties of allyl luciferin in combination with various luciferases
a
Apparent Km [µM]
Apparent Vmax
Luciferase
[×108photons/sec]
Fluc
Eluc
SLG
SLO
SLR
23.8±6.9
18.8±6.4
40.6±8.4
10.2±4.8
12.5±7.2
7.69
6.93
0.0725
0.0292
0.0751
a Vmax values are within 5% error
similar red-shifted behaviour for sterically hindered luciferins
(e.g. alkyn- or halogen-substituted) with Fluc.24,25The results
obtained in this study suggested that allyl modification at the
C-7` position influences the hydrophobic environment of the
active site of Fluc, only. While continuous emission was
observed for D-luciferin in all luciferases, the emission of 7`-
AllylLuc with Fluc gradually decreased with time, with the
emission intensity reaching half of its original value 12 seconds
after the start of the reaction (Fig. 2b). This decrease in
bioluminescence intensity with Fluc may be due to inhibition
by the reaction product. Surprisingly, the emission intensity
with ELuc was the highest among all the luciferases, and the
maximum emission intensity was sustained (Fig. 2b). Since
bioluminescence imaging generally requires long-term
exposure, prolonged luminescence is a very important
characteristic for both in vitro and in vivo imaging.
To further understand the enzymatic reaction, the apparent
Michaelis constants Km (defined as the concentration at half of
the maximum reaction rate) and the maximum velocities Vmax
for 7`-AllylLuc with each luciferase were evaluated. The initial
luminescence intensity was measured as a function of luciferin
concentration (Fig. S6). All 7`-AllylLuc - luciferase pairs showed
typical Michaelis-Menten plots. The Km and Vmax values were
calculated from the Lineweaver-Burk plots. Little variation
among Km values was observed for allyl luciferin (Table 1). On
the other hand, there were notable differences in the Vmax
values, which displayed a strong correlation with the emission
intensity shown in Fig. 2. Moreover, the Vmax values were
3~100-fold lower compared with that of the D-luciferin - Fluc
pair, the most commonly used luciferin-luciferase pair (data
not shown). In order to further discuss the difference in
luminescence efficiency between
D-luciferin and 7`-AllylLuc,
bioluminescence quantum yield measurements with Fluc and
Eluc were performed (Fig. S7). They revealed that 7`-AllylLuc
has about 10% of the luminous efficiency with both luciferases
Fig. 3 Light emission from luciferase-expressing COS-7 cells treated with 7`-AllylLuc: a)
total intensities of dose-response emission; b) time courses at 100 µM substrate; Error
bars represent the standard deviation of 4 experiments; c) imaging of luciferase-
expressing COS-7 cells treated with D-luciferin or 7`-AllylLuc.
(Fluc and Eluc) compared to the natural substrate D-luciferin
(Table S1). These results suggest that while allyl modification
at the C-7` position enables enzyme recognition, it may
interfere with the catalysis necessary to emit light. Steric
hindrance by modification with an allyl group may prevent
effective luminescence processing of allyl luciferin, like it has
been previously reported in the case of brominated
luciferins.25
concentrations. While the light output from 7`-AllylLuc was
weaker compared to D-luciferin (Fig. 3, Fig. S8 and S9), a dose-
dependent response was observed (Fig. 3a). No detectable
signal could be obtained at low concentrations (5 μM) of
luciferin, presumably due to the low cell membrane
permeability of the luciferins. Surprisingly, specific light
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7
8
9
V. R. Viviani, E. J. H. Bechara and Y. Ohmiya, Biochemistry
(Mosc.), 1999, 38, 8271–8279.
B. R. Branchini, T. L. Southworth, N. F. Khattak, E. Michelini and
A. Roda, Anal. Biochem., 2005, 345, 140–148.
Y. Nakajima, T. Yamazaki, S. Nishii, T. Noguchi, H. Hoshino, K.
Niwa, V. R. Viviani and Y. Ohmiya, PLoS ONE, 2010, 5, e10011.
emission was observed with Eluc-expressing cells, only (Fig. 3).
Furthermore, the 7`-AllylLuc-based light emission from Eluc-
expressing cells was sustained like in the case of the native D-
luciferin emitting system and lasted over 100 minutes, which is
a desirable property for bioluminescence applications, as
already noted before. Cell viability evaluation by a standard
MTT assay revealed that there was no significant cytotoxicity
of 7`-AllylLuc even after treatment with a high dosage of 7`-
AllylLuc (500 μM) for 24 h (Fig. S10). Despite 7`-AllylLuc having
10 E. H. White, F. McCapra and G. F. Field, J. Am. Chem. Soc., 1963,
85, 337–343.
11 E. H. White, H. Wörther, G. F. Field and W. D. McElroy, J. Org.
Chem., 1965, 30, 2344–2348.
weaker emission intensity,
a significant benefit of this
12 E. H. White, E. Rapaport, T. A. Hopkins and H. H. Seliger, J. Am.
Chem. Soc., 1969, 91, 2178–2180.
analogue is its high luciferase specificity for Eluc, making it a
potentially promising tool for multi-target imaging.
13 N. Lembert, Biochem. J., 1996, 317, 273–277.
14 S. Ioka, T. Saitoh, S. Iwano, K. Suzuki, S. A. Maki, A. Miyawaki, M.
Imoto and S. Nishiyama, Chem. - Eur. J., 2016, 22, 9330–9337.
15 D. C. McCutcheon, M. A. Paley, R. C. Steinhardt and J. A.
Prescher, J. Am. Chem. Soc., 2012, 134, 7604–7607.
16 C. C. Woodroofe, P. L. Meisenheimer, D. H. Klaubert, Y. Kovic, J.
C. Rosenberg, C. E. Behney, T. L. Southworth and B. R. Branchini,
Biochemistry (Mosc.), 2012, 51, 9807–9813.
17 H. Takakura, R. Kojima, T. Ozawa, T. Nagano and Y. Urano,
ChemBioChem, 2012, 13, 1424–1427.
18 S. Iwano, R. Obata, C. Miura, M. Kiyama, K. Hama, M. Nakamura,
Y. Amano, S. Kojima, T. Hirano, S. Maki and H. Niwa,
Tetrahedron, 2013, 69, 3847–3856.
In conclusion, a novel luciferin analogue modified with an
allyl group at the C-7` position was successfully synthesized. In
cellular imaging, this 7`-AllylLuc displayed
a luciferase-
selective bioluminescence signal in combination with Eluc,
which is one of the commercially available beetle luciferases
from Pyrearinus termitilluminans. Studies are now in progress
to develop artificial beetle luciferases that display stronger
bioluminescence emission with 7`-AllylLuc compared to that
of D-luciferin, and to apply its selective response to
bioluminescence-based reporter assays.
In addition, besides of being
a promising luciferase
19 C. Miura, M. Kiyama, S. Iwano, K. Ito, R. Obata, T. Hirano, S.
Maki and H. Niwa, Tetrahedron, 2013, 69, 9726–9734.
20 K. A. Jones, W. B. Porterfield, C. M. Rathbun, D. C. McCutcheon,
M. A. Paley and J. A. Prescher, J. Am. Chem. Soc., 2017, 139,
2351–2358.
21 K. R. Harwood, D. M. Mofford, G. R. Reddy and S. C. Miller,
Chem. Biol., 2011, 18, 1649–1657.
22 S. T. Adams, D. M. Mofford, G. S. K. K. Reddy and S. C. Miller,
Angew. Chem. Int. Ed., 2016, 55, 4943–4946.
23 T. Nakatsu, S. Ichiyama, J. Hiratake, A. Saldanha, N. Kobashi, K.
Sakata and H. Kato, Nature, 2006, 440, 372–376.
24 R. C. Steinhardt, J. M. O’Neill, C. M. Rathbun, D. C. McCutcheon,
M. A. Paley and J. A. Prescher, Chem. - Eur. J., 2016, 22, 3671–
3675.
25 R. C. Steinhardt, C. M. Rathbun, B. T. Krull, J. M. Yu, Y. Yang, B.
D. Nguyen, J. Kwon, D. C. McCutcheon, K. A. Jones, F. Furche
and J. A. Prescher, ChemBioChem, 2017, 18, 96–100.
substrate by itself, the terminal olefin function of the allyl
group is available as a linker for further covalent modifications.
It is convertible to aldehyde groups by Lemieux-Johnson
oxidation, which can then easily react with various functional
groups.17 Therefore, allyl group introduction into firefly
luciferin enables further useful possibilities, such as the
development of various analogues and labelling applications.
It is believed that this new synthetic luciferin will contribute
to the expansion of bioluminescence imaging applications
both in vitro and in vivo.
This study was supported by a Grant-in Aid for Scientific
Research (S) (Grant No. 24225001) to K.S. from the Japan
Society for the Promotion of Science (JSPS).
Conflicts of interest
There are no conflicts of interest to declare.
Notes and references
1
2
3
4
5
J. A. Prescher and C. H. Contag, Curr. Opin. Chem. Biol., 2010, 14,
80–89.
S. T. Adams and S. C. Miller, Curr. Opin. Chem. Biol., 2014, 21,
112–120.
M. A. Paley and J. A. Prescher, Med Chem Commun, 2014, 5,
255–267.
P. Colepicolo-Neto, C. Costa and E. J. H. Bechara, Insect
Biochem., 1986, 16, 803–810.
K. Niwa, Y. Ichino, S. Kumata, Y. Nakajima, Y. Hiraishi, D. Kato, V.
Viviani and Y. Ohmiya, Photochem. Photobiol., 2010, 86, 1046–
1049.
6
V. R. Viviani, A. C. R. Silva, G. L. O. Perez, R. V. Santelli, E. J. H.
Bechara and F. C. Reinach, Photochem. Photobiol., 1999, 70,
254–260.
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