Bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution

Article abstract of DOI:10.1039/c4cc06886f

Three novel coelenterazine (CTZ) derivatives with extension at the C-6 position of the imidazopyrazinone structure show significant bioluminescence emission with known renilla luciferase variants, indicating a promising method to develop CTZ derivatives w

Full text of DOI:10.1039/c4cc06886f

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Bioluminescent coelenterazine derivatives with
imidazopyrazinone C-6 extended substitution†
Cite this:DOI: 10.1039/c4cc06886f
Ryo Nishihara,a Hideyuki Suzuki,b Emi Hoshino,a Sakura Suganuma,b
Moritoshi Sato,b Tsuyoshi Saitoh,c Shigeru Nishiyama,c Naoko Iwasawa,a
Daniel Citterioa and Koji Suzuki*a
Received 1st September 2014,
Accepted 11th November 2014
DOI: 10.1039/c4cc06886f
www.rsc.org/chemcomm
Three novel coelenterazine (CTZ) derivatives with extension at
the C-6 position of the imidazopyrazinone structure show signifi-
cant bioluminescence emission with known renilla luciferase
variants, indicating a promising method to develop CTZ derivatives
with superior optical properties compared to hitherto reported
compounds.
Bioluminescence-based assays such as gene reporter assays in cell
cultures and in vivo imaging are known for their low background
signals and high sensitivity.1 The mechanism relies on an enzymatic
oxidation reaction involving a bioluminescent substrate (luciferin)
and an enzyme (luciferase).
Fig. 1 The structure of native CTZ and CTZ derivatives.
The luciferin most widely applied in imaging assays is the firefly
luciferin, which emits light at a relatively long wavelength
(560 nm).2–5 Unfortunately however, the process leading to light mostly unknown.15–17 In fact, most of the reported CTZ derivatives
emission of firefly luciferin requires the presence of Mg2+ ions and fail to emit bioluminescence, since their structural modifica-
ATP as co-factors, which potentially leads to complex assay protocols tions prevent their enzymatic recognition.5–9 The luminescence
in bioanalysis. For this reason, the Mg2+ and ATP independent capacity of CTZ is due to its imidazopyrazinone structure. In
renilla luciferase (Rluc) enzyme in combination with coelenterazine previously published work, the effects on the bioluminescence
(CTZ) (Fig. 1a) as the substrate has recently been used as a versatile properties of substitution at the C-2, C-5, C-6 and C-8 positions
reporter protein.6 However, the Rluc/CTZ pair results in relatively of the imidazopyrazinone core have been investigated.9,10,14,18,19
short wavelength emission in the blue spectral region (480 nm) with
Although it is generally said that the effect of substitution at
a low intensity compared to other bioluminescent systems.7,8 To the C-2 position on enzymatic recognition is relatively low, the
overcome these limitations, there is a lot of interest in developing benzyl group at the C-2 position is a prerequisite for the lumines-
new CTZ derivatives.9–14 But the design of novel CTZ derivatives cence of imidazopyrazinone derivatives.19 Few C-2 CTZ derivatives
resulting in better optical characteristics is challenging, since the show luminescence properties superior to those of native CTZ,
detailed enzymatic recognition mechanism of the Rluc system is still except in combination with aequorin. CTZ derivatives modified at
the C-8 position have also been reported.9 A compound having a
styryl group at the C-8 position (Fig. 1b) showed approximately
120 nm red-shifted chemiluminescence emission compared to that
a Department of Applied Chemistry, Faculty of Science and Technology,
Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522,
of the native CTZ, but this structural change resulted in negligibly
Japan. E-mail: suzuki@applc.keio.ac.jp
low bioluminescence in combination with Rluc.20
b Department of Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
c Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba,
Meguro-ku, Tokyo 153-8902, Japan
Different types of CTZ derivatives (e-CTZ and v-CTZ) have been
obtained by forming a bridge between the C-5 carbon and the
( p-hydroxy)-phenyl substituent at the C-6 position of the native
CTZ.14,21 This modification leads to more planar and rigid
molecular structures with increased bioluminescence emission,
however at the cost of molecular stability.21
† Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedures and spectral characterization of new compounds; additional information
on chemiluminescence and bioluminescence properties as noted in the text; and
summarized luminescence properties. See DOI: 10.1039/c4cc06886f
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As mentioned above, native CTZ has a ( p-hydroxy)-phenyl
In the case of BL, the spectral location of the emission band
group in the C-6 position. Some CTZ derivatives with alternative depends not only on the chemical structure of the CTZ derivative,
substituents at this position have also been reported.12,13,22,23 but to a large extent on its interactions with proximal amino acid
However, most of these studies have focused on the chemi- residues in the luciferase enzyme,16 which remain to a large degree
luminescence properties.
still unknown. Upon oxidation in the presence of Rluc, native CTZ
There are only a few studies on the C-6 substituent effects on is converted into the light emitting excited state coelenteramide,
enzymatic recognition and on the bioluminescence properties with its emission wavelength depending on the protonation state.
of the resultant CTZ derivatives.14 To fill this gap, we have It is hypothesized that the red-shifted emission of native CTZ and
decided to further investigate the influence of substituents at 6-pi-OH-CTZ with Rluc8.6 can be attributed to the excited state
the C-6 position of native CTZ on the chemiluminescence (CL) coelenteramide existing in its deprotonated pyrazine anion form
and bioluminescence properties (BL). Compared to BL, the inside the enzymatic pocket. This assumption is supported by the
measurement of CL allows us to study the structure-related fact that CTZs having a hydroxyl group on the C-6 substituent
basic luminescence properties independent of any possible (native CTZ, 6-pi-OH-CTZ) show a red-shift in their CL emissions
luciferin-luciferase mismatch.24–26
upon addition of a base to the sample solution (Fig. S2 and
As the first new derivative, the styryl-substituted compound Table S1, ESI†). Based on a previous report, the spectral shoulders
6-pi-OH-CTZ was successfully synthesized (Fig. 1d). Its CL spectrum near 400 nm observed in Fig. 2b are assumed to originate from the
is shown in Fig. S1 (ESI†). In contrast to the significantly red-shifted neutral species of coelenteramide15 due to incomplete deprotona-
C-8 styryl-substituted compound,20 the spectral similarity between tion of the hydroxyl group on the C-6 substituent.
6-pi-OH-CTZ and native CTZ shows that the C-6 substituent has
As the next compound, 6-pi-H-CTZ (Fig. 1e) was designed and
little influence on the CL emission wavelength. This result is synthesized to evaluate the effect of the absence of the hydroxyl
not really surprising, since it has been previously reported that group in the C-6 substituent on the CL and BL properties. The
even the complete absence of a substituent at C-6 still results in a measurement of the CL spectrum of 6-pi-H-CTZ (Fig. S1, ESI†) was
CL emission peak at around 450 nm.27 This finding has been carried out under the same conditions as in the case of 6-pi-OH-CTZ.
generally attributed to a dihedral angle twist between the imidazo- Also for this substitution pattern, the emission peak of 6-pi-H-CTZ
pyrazinone core and the substituent at the C-6 position in various was found at 478 nm, almost identical to the native CTZ.
CTZ derivatives, which prevents an effective extension of p-electron
It has been reported that the replacement of the p-hydroxyl
group on the 6-phenyl substituent of native CTZ can drastically
conjugation from the imidazopyrazinone core.28
Next, the enzymatic recognition-dependent BL properties of decrease the capacity of BL emission.11 Fig. 3 shows the results
6-pi-OH-CTZ were investigated using native renilla luciferase of BL measurements with 6-pi-H-CTZ. Emission was observed
(Rluc) and the widely known Rluc8 and Rluc8.6 variants.15 in combination with the Rluc8 and Rluc8.6 luciferases (Fig. 3a).
Fig. 2 shows a comparison of the BL intensities and spectra The intensity of 6-pi-H-CTZ with Rluc8.6 is approximately 41% of
of 6-pi-OH-CTZ and native CTZ. Fig. 2a indicates that 6-pi-OH- that of native CTZ/Rluc. The BL spectra recorded in the presence of
CTZ is recognized by Rluc8 and Rluc8.6, but not by Rluc. The Rluc8 and Rluc8.6 are both significantly blue-shifted compared to
BL intensity of the 6-pi-OH-CTZ/Rluc8 pair is 44% of that of the those of native CTZ (Fig. 3b). This different BL spectral behaviour in
native CTZ/Rluc combination. On the other hand, the intensity comparison with 6-pi-OH-CTZ clearly indicates that the p-hydroxyl
of 6-pi-OH-CTZ with Rluc8.6 is 123% of that of the CTZ/Rluc pair group of the C-6 substituent is involved in the enzymatic inter-
and therefore it is a relatively bright derivative. As for the BL action of CTZs with the Rluc series luciferases. The absence of that
spectra with Rluc8.6, the emission wavelengths of 6-pi-OH-CTZ hydroxyl group prevents the formation of the pyrazine anion upon
and native CTZ are very similar (Fig. 2b). However, with Rluc8, enzymatic oxidation, most reasonably resulting in the neutral
6-pi-OH-CTZ shows approximately 50 nm red-shifted emission coelenteramide, which emits at a short wavelength. With the two
compared to that of CTZ (Fig. 2b). As a result, it can be CTZ derivatives 6-pi-OH-CTZ and 6-pi-H-CTZ, it has been demon-
concluded that CTZ derivatives with a p-conjugated substituent strated that compounds having a styryl group at the C-6 position
at the C-6 position can show significant BL emission with the show significant BL emission in combination with the known Rluc
known Rluc variants, Rluc8 and Rluc8.6.
variants, Rluc8 and Rluc8.6. This is a clear indication that CTZ
Fig. 2 Luminescence properties of 6-pi-OH-CTZ: (a) BL intensities; (b) BL
Fig. 3 Luminescence properties of 6-pi-H-CTZ: (a) BL intensities; (b) BL
spectra with Rluc8 and Rluc8.6.
spectra with Rluc8 and Rluc8.6.
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derivatives obtained by replacing the original ( p-hydroxy)-
phenyl substituent at the C-6 position undergo enzymatic
recognition by Rluc8 and Rluc8.6. To the best of our knowledge,
the styryl-groups are the largest examples of C-6 substituents
reported so far, with the resulting CTZ derivatives still showing
significant BL emission.
To further evaluate the possibility of extending the substituent
at the C-6 position, 6-pi-Ph-CTZ having a bulky phenyl group in the
para position of the 6-styryl group was designed and synthesized
(Fig. 1f). Interestingly, the CL spectrum of 6-pi-Ph-CTZ (Fig. S1,
ESI†) showed a 50 nm red-shifted emission compared to that of
native CTZ. This seems to be in contradiction to the dihedral angle
twist mentioned earlier. At this time, it is only possible to assume
that the electronic properties of the additional phenyl group (e.g. its
weak inductive electron withdrawing effect) result in an extension
of the conjugated p-electron system by a reduction of the dihedral
twisting at the C-6 position. The results of bioluminescence
measurements are shown in Fig. 4a and b.
Fig. 5 Comparison of BL properties: (a) BL intensity; (b) BL spectra with
Rluc8 and (c) BL spectra with Rluc8.6.
A comparison of BL intensities (Fig. 4a) shows that 6-pi-Ph-CTZ
is recognized by Rluc8 and Rluc8.6, and in combination with with emission kinetics and half-life times of an order similar to
Rluc8.6 its intensity reaches 94% of that of the native CTZ/Rluc native CTZ (Fig. S4–S8, Table S2, ESI†).
pair. This result indicates that the structural modification at the C-6
An example of a commercially available CTZ derivative,
position has little influence on the enzymatic recognition by Rluc8 emerging from structural modifications at both C-2 and C-6
and Rluc8.6. The corresponding bioluminescence spectra (Fig. 4b) (missing hydroxyl substituent) positions of the imidazopyrazinone
show the emission peak of 6-pi-Ph-CTZ at 418 nm with Rluc8.6. core, is the emission wavelength blue-shifted DeepBlueCt
This is again significantly blue-shifted in comparison to native CTZ, (Fig. 1c).29 DeepBlueCt is used as a donor in BL resonance energy
presumably because of the absence of a deprotonatable hydroxyl transfer (BRET)-based applications with green fluorescent protein
group. As a result, 6-pi-Ph-CTZ is the brightest short wavelength (GFP) as the acceptor, because its emission peak at around 400 nm
emitting CTZ derivative reported so far.
minimally interferes with the emission of the GFP acceptor.
All of the three CTZ derivatives evaluated in this study having a Typical applications are BRET studies to image protein–protein
styryl group on the C-6 position of the imidazopyrazinone core interactions.30 However, the BL emission intensity of DeepBlueCt
showed weak BL emission with Rluc8, but significantly stronger is only 3.7% compared to that of native CTZ. Since the BL emission
emission with Rluc8.6. In contrast to native CTZ (480 nm in Rluc8 of 6-pi-H-CTZ and of DeepBlueCt is in a similarly short wavelength
and 526 nm in Rluc8.6), the three new derivatives show no range, a comparison of the BL properties of the two compounds
differences in the BL emission wavelength between Rluc8 and was performed (Fig. 5). In terms of BL intensities, 6-pi-H-CTZ
Rluc8.6. Therefore, it can be assumed that C-6 styryl-substituted and 6-pi-Ph-CTZ with Rluc8.6 showed approximately 11-fold and
CTZ derivatives interact with different amino acid residues com- 25-fold stronger emission than DeepBlueCt with Rluc8 (Fig. 5).
pared to native CTZ. A further hint in this direction is the fact that Therefore, 6-pi-H-CTZ and 6-pi-Ph-CTZ are promising bright
native CTZ also emits BL in combination with other related blue-shifted CTZ derivatives.
enzymes, such as Gaussia luciferase, whereas 6-pi-H-CTZ and
6-pi-OH-CTZ are selectively recognized by the Rluc8 and Rluc8.6 the light emitting species are very high. Although many details
variants only (Fig. S3, ESI†). still remain unknown, the emission wavelength of native CTZ
In BL, the environmental effects of the enzyme active site on
Measurements of the bioluminescence emission time profiles with various luciferases is significantly altered by the surrounding
revealed that all three CTZ derivatives show flash-type luminescence environment determined by amino acid residues due to factors
such as hydrophobicity and pH changes.
In this work, three types of novel CTZ derivatives with styryl
substituents at the C-6 position have been successfully developed.
All of them showed strong BL emission in combination with the
Rluc8 and Rluc8.6 renilla luciferase variants, with brightest
emission observed in the case of the 6-pi-Ph-CTZ/Rluc8.6 pair.
With 11-fold and 25-fold stronger emission at comparable
wavelengths and similar bioluminescence emission half-life
times (Fig. S6–S8, Table S2, ESI†), 6-pi-H-CTZ and 6-pi-Ph-CTZ
are useful bright blue-shifted alternatives to DeepBlueCt, which
is commonly applied in BRET-based assays in combination
with GFP.
Fig. 4 Luminescence properties of 6-pi-Ph-CTZ: (a) BL intensities; (b) BL
spectra with Rluc8 and Rluc8.6.
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