Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties

Article abstract of DOI:10.1039/C9OB00459A

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Full text of DOI:10.1039/C9OB00459A

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Goyard, V. Hervin, G. Gagnot, R. Baatallah, Y. Jacob, T. Rose and Y. L. Janin, Org. Biomol. Chem., 2019,
DOI: 10.1039/C9OB00459A.
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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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DOI: 10.1039/C9OB00459A
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Gram-Scale Synthesis of Luciferins Derived from Coelenterazine
and Original Insights in Their Bioluminescence Properties
Eloi P. Coutant,a Sophie Goyard,b Vincent Hervin,a Glwadys Gagnot,ac Racha Baatallah,a Yves
Received 00th January 20xx,
Accepted 00th January 20xx
Jacob,d Thierry Rose*b and Yves L. Janin*a
DOI: 10.1039/x0xx00000x
An original gram-scale synthesis of O-acetylated forms of
coelenterazine, furimazine or hydroxy-bearing analogues of these
luciferins is described. The comparisons over two hours of their
bioluminescence, using the nanoKAZ/NanoLuc luciferase, is
providing remarkable insights usefull for the selection of a
substrate adapted for a given application.
OH
O
O
N
N
N
N
HN
NH2
NH
N
H
N
H
HN
Bioluminescence is based on the combination of at least
oxygen, a small chemical cofactor and an enzyme. As depicted
in scheme 1, extensive research,1 led to the discovery that many
sea-dwelling bioluminescent species2, 3 are using coelenterazine
(CTZ, 1) or varguline (2) and harbor a large variety of luciferases
or photoproteins (which are calcium-dependent luciferases) to
produce light with these. Across the years, because of the many
uses of bioluminescence-based tools in life sciences,4-10
attempts were made to improve the signal intensity, duration
and/or the emission wavelength. This started with the isolation
of a wide range of coelenterazine-using luciferases and their
combination with analogues of coelenterazine (1). It then
moved to the analysis of the effect of luciferase mutations on
the bioluminescence properties and the best results obtained
so far were achieved by the association of mutated luciferases
and luciferins analogues.11-25 It is the combinations of an
extensively mutated form of the catalytic subunit of Oplophorus
gracilirostris luciferase (nanoKAZ/NanoLuc)20, 21 and furimazine
(3),20 or bisdeoxycoelenterazine (bis-CTZ, 4),21 which appears to
be the current state of the art in regard with signal intensity and
duration.
HO
1
2
O
O
O
N
N
N
N
N
H
N
H
3
4
O
N
NH2
R2
Main
accesses
EtO
O
+
R1
N
R3
7
R2
N
O
O
O
R2
6
or
N
8
O
N
Cl
R1
N
H
R3
R2
NH2
+
This
work
EtO
R1
N
R3
5
9
10
Scheme 1. Structures of coelenterazine (1), varguline (2), furimazine (3), and
bisdeoxycoelenterazine (4) and retrosynthesis of imidazo[1,2-a]pyrazine-3(7H)-ones
a. Unité de Chimie et Biocatalyse, Institut Pasteur, UMR 3523, CNRS, 28 rue du Dr.
Roux, 75724 Paris cedex 15, France.
Concerning the chemistry of these luciferins, they are oxygen-
sensitive and readily decompose in solution especially in the
presence of a base, or upon light exposure.26 As depicted above,
many27 if not all28-30 the reported preparations of imidazo[1,2-
a]pyrazin-3(7H)-ones 5 are requiring an aminopyrazine (6) prior
to the construction of the imidazole ring via a condensation with
α-ketoesters (7) or α-ketoaldehydes (8). Recent improvements
have extended this access to original luciferins24, 26 but to avoid
some of its inherent limitations, we focused on an alternative
b. Center for Innovation and Technological Research, Institut Pasteur, 25 rue du Dr.
Roux, 75724 Paris cedex 15, France.
c. Université Paris Descartes, Sorbonne Paris Cité, 12 rue de l'Ecole de Médecine,
75006 Paris, France.
d. Unité de Génétique Moléculaire des Virus à ARN, Institut Pasteur, UMR 3569,
CNRS, 28 rue du Dr. Roux, 75724 Paris cedex 15, France.
Electronic Supplementary Information (ESI) available: All experimental details for
the synthesis of the (pro)luciferins, their deprotection, and the subsequent
biochemical characterizations of their bioluminescence are provided. See
DOI: 10.1039/x0xx00000x
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initially explored on a model compound.31 This path not only Indeed, in our cases, it is only when removing the solvent that
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DOI: 10.1039/C9OB00459A
avoids the use of the non-trivial intermediates 7-8, but it also the 1,4 adducts 13a-c were formed. The use of zinc and
offers, via a key N-arylation of halogenopyrazines (9), the hydrochloric acid in dioxane for their reduction provided an
recourse to a wide range of the far more available α-amino access to the diamines 14a-c including a compatibility with the
esters (10).32-34 As depicted in scheme 2, an original preparation benzyloxy groups of 13b-c. Their cyclization to give the
of chloropyrazines 9a-c was achieved starting with a 1,4- (separable) piperazinone diastereoisomers 15a-c was then
addition reaction between β-nitrostyrenes 11a-c and achieved with heat. The previously unreported use of sulfur as
phenylalanine ethyl ester (12).
an oxidant was initially essential for their aromatization into the
corresponding hydroxypyrazines 16a-c. Later on, we found an
alternative via an original dehydration of the N-oxide 17a-b
(obtained by the peroxyacetic acid treatment of 15a-b) using
either heat or more preferably sodium hydroxide. Finally, from
the hydroxypyrazines 16a-c, hot phenylphosphonic dichloride42
was essential to prepare the chloropyrazines 9a-c, and this
reagent could also be used to directly transform, for instance,
N-oxide 17a into the chloropyrazine 9a. This was followed by
NO2
R1
CO2Et
NO2
CO2Et
Ph
i
+
H2N
N
R1
H
Ph
11a-c
12
13a-c
H
N
H2N
O
CO2Et
ii
iii
R1
N
R1
N
H
H
the key Buchwald-Hartwig palladium-catalyzed N-arylation of
Ph
Ph
34
the readily available33,
α-amino esters 10A-C by
14a-c
15a-c
chloropyrazines 9a-c. Starting from related precedents,43-48 it
quickly turned out that a mild temperature was required. The
best conditions we found, 60 °C in acetonitrile with cesium
carbonate for 12 hours using BINAP and palladium(II) acetate,
led to the N-arylesters 18 in 69-90% yields. For the next step,
we observed that the inherent instability of the target luciferins
5aB limited its purification to a precipitation. To avoid this, we
prepared the far more stable O-acetylated derivatives 20 in one
pot from the N-arylesters 18 via the acid salts 19, generated in
situ, and an ensuing treatment with an excess of acetic
anhydride. These pro-luciferins turned out to be stable enough
to withstand a chromatography but, even better, a simple
recrystallization provided compounds 20aA-B in up to grams
amount. For the synthesis of the phenol-bearing luciferins, a
catalytic hydrogenation of O-benzyl-bearing compounds 20bA-
bC and 20cB-cC provided the corresponding O-acetylated
luciferins 20dA-dB, 20dD, 20eB and 20eD. Then, as seen by
LC/MS (supporting information, figure S1), a treatment of these
O-acetylated luciferins 20 with a mixture of hydrochloric acid,
ethanol and DMSO at 50 °C provided concentrated solutions of
the pure luciferins 5 which could be used immediately, upon a
dilution in the relevant buffer, or stored at low temperature. As
depicted in figure 2, and described in the supporting
information section, the bioluminescence properties of these
luciferins were then studied using a purified recombinant
nanoKAZ/NanoLuc luciferase. In comparison with the very low
intensity of coelenterazine (1), or for that matter
N
OH
N
Cl
iv
v
R1
N
R1
N
Ph
Ph
16a-c
9a-c
v
vii
H
N
O
a: R1 = C6H5-
b: R1 = 4-BnOC6H4-
c: R1 = 3-BnOC6H4-
vi
15a-b
R1
N+
O-
Ph
17a-b
EtO2C
N
NaO2C
R2
EtO2C
R2
R2
NH2
NH
Ph
N
NH
vii
viii
9a-c
10A-C
N
R1
N
R1
Ph
18aA-B
18bA-C
18cB-C
19aA-B
19bA-C
19cB-C
R2
R2
AcO
O
ix
xi
N
N
N
N
N
R1
R1
N
H
Ph
Ph
20aA-B
20bA-C
20cB-C
x
5aA-B
20dA-B, 20dD
20eB, 20eD
“isocoelenterazine”
(5eD),
furimazine
(3)
and
5dA-B, 5dD
5eB, 5eD
bisdeoxycoelenterazine (4) were, as previously reported,20, 21
providing vastly improved bioluminescence signals lasting at
least two hours. Interestingly, the two monohydroxy-bearing
analogues h-coelenterazine (5dA) and 5dB led to at least twice
more intense signals but which lasted only minutes (figure 2B).
Such initial intensity has actually been reported before for h-
coelenterazine (5dA).21 On the other hand, the isomeric mono
hydroxy-bearing compound 5eB displayed a far more stable
bioluminescence profile pretty much identical with the one
observed for furimazine (3). With all these luciferins, the light
intensity decreased with time: rather quickly for the “flash” and
a: R1 = C6H5
5aA is 4 (bis-CTZ)
5aB is 3 (furimazine)
5dD is 1 (CTZ)
b: R1 = 4-BnOC6H4 A: R2 = C6H5
c: R1 = 3-BnOC6H4 B: R2 = 2-furyl
d: R1 = 4-HOC6H4 C: R2 = 4-BnOC6H4
e: R1 = 3-HOC6H4 D: R2 = 4-HOC6H4
Scheme 2. i: neat. ii: Zn, H3O+, Cl-, dioxane. iii: 130 °C, neat. iv: S8, 1,3-Cl2C6H4,
reflux. v: PhPOCl2, 100 °C. vi: AcOOH, AcOEt. vii: chlorobenzene, reflux or NaOH,
EtOH, reflux. . vii: Cs2CO3, Pd(OAc)2, BINAP, MeCN, 60 °C. viii: NaOH, THF, 20 °C. ix: Ac2O,
20 °C. x: H2, Pd/C, AcOEt, AcOH, EtOH. xi: EtOH, DMSO, H3O+, Cl-, 50 °C.
2 | J. Name., 2012, 00, 1-3
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much more slowly for the “glow” substrates. For the “flash” Accordingly, the enzyme is irreversibly inactivatedVbieywaArrtieclaecOtniloinne
DOI: 10.1039/C9OB00459A
substrates, luciferins 5dA and 5dB, the decrease fitted with a product in all the cases with a constant (k ) dependent on the
inact
first order equation. Adding further substrate after losing 90% substrate used. Of note is that when all the substrate has been
of the initial light emission intensity did not, at best, produce consumed, the area below these curves is providing a molecules
any changes and could even causes a decrease of the remaining consumed per RLU produced ratio (see table 1). Concerning the
signal intensity. On the other hand, as depicted in figure 2C, kinetics of these reactions, they are fitting with a Michaelis-
adding instead the same amount of enzyme used at the start Menten model, if we assume that the number of detected
led to a recovery of the signal. For the others substrates, this photons per consumed substrate molecule is constant
enzyme death was less pronounced but took place anyway (see whatever the substrate concentration (see the supporting
supporting information).
information for a discussion). The KM and Vmax values were
computed considering: 1) the luciferins (S) as the limiting
substrates, O2 as saturating substrate 2) an inhibition of the
enzyme E by excess of substrate through the binding of a second
substrate (ESS) on the Michaelis’ complex (ES) with the
dissociation constant KI and 3) a stochastic inactivation of the
enzyme (E*) with the kinetic constant kinact. As seen in table 1
and figure 3, coelenterazine (1) has a very high kcat but a poor
photon emission efficiency. Interestingly, furimazine (3) and its
isomer 5eB are the most efficient photon emitter per substrate
molecule catalysis with the same kcat but furimazine (3) is
somehow providing a longer life time of active enzyme (low
kinact). All substrates are sensitive to substrate concentration
beyond the KM, but two luciferins, coelenterazine (1) and 5eD,
are more affected than others as seen with their low
dissociation constants (KI). Of note is that the strong light
intensity produced by the catalyzed oxidation of 5dB in a very
short time (flash) does not mean the production of a high level
of light intensity cumulated in two hours (ΣI) and the very long
half-life (tI/2) with low light intensity (glow) provided by
compound 5eD neither. Among these substrates, furimazine (3)
is, so far, providing the best compromise. Also quite unexpected
is the reason behind the fact that the natural substrate
coelenterazine (1) leads to a signal two orders of magnitudes
less intense than furimazine (3). Indeed, it is not because of a
lesser catalytic activity but it is mostly due to a pretty much
counter intuitive lesser number of photons detected by luciferin
consumed.
A
O
O
O
N
N
N
N
N
H
N
H
HO
HO
OH
5dA
5dB
O
O
O
N
N
N
N
HO
HO
N
N
H
H
5eD
5eB
Table 1. Kinetic parameters for each substrate.
3
4
5dA
5dB
1
5eB 5eD
Imax
(106 RLU s-1)
1.69
3.28
2.05
3.11
0.23
1.98 0.10
t½ (min)
74.29 19.34
84.98 80.85
3.22
5.61
3.71
0.33
11.57
3.45
20.96 32.27 148
6.29 62.97 6.04
I (106 RLU)
K’M (10-6 M)
k’cat
3.40
2.33
8.8
4.23 7.02
2.33 0.17
7002
1.80
3.50
2.32
3.60
0.44
(1018 RLU s-1)
Molecules/RLU 1775
4581 12067 4463 101169 1804
0
KI (10-6 M)
KM (10-6 M)
104.94 115.21 140.55 101.64 22.48 84.02 35.42
2.22
106
1.5
3.02
534
5.2
5.90
932
425
3.88
535
375
6.80
1500
3.8
3.30 2.94
kcat (mol.s-1
Figure 2. Bioluminescence signals of the luciferins
5 using recombinant
140 362
-1
nanoKAZ/NanoLuc. (A) Luciferin structures; compounds 1, 3, and 4 are depicted in figure
1. (B) Light intensity in RLU s-1 plotted vs. time over two hours, the insert zooms on the
first two minutes of the reaction. (C) Bioluminescence profiles of compound 5dB at
different concentrations along with the repeated addition (arrows) of luciferase.
.molE
)
kinact (10-4 s-1)
4.5
3.1
An alternative relative depiction of some of these variables is provided figure 3.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Figure 3. Depiction of the maxima of intensities (Imax), integrated signals (ΣI), and half-
Figure 3 is probably providing a better visual representation of
these differences. In comparison with the central equidistant
triangle representing three characteristics of furimazine (3):
maximum intensity (Imax), the sum of the signal (ΣI), and half-
life of the signal over two hours (t1/2), the values for the other
substrates can vary widely. Accordingly, the next stage of our
research will be to find out how specific structural features of
the luciferins have an influence on these characteristics and
more importantly, can such changes lead to even better
luciferins.
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time durations (t1/2) for all the luciferins, relative to furimazine (3) represented in light
DOI: 10.1039/C9OB00459A
grey.
The kinetic analyses of light emission presented here are
providing rather unexpected insights for distinct applications.
The glowing property of some substrates is appropriate for high
throughput in vitro bioassays and long imaging dynamics in vivo
or in cellulo, whereas the flashier profile of other is appropriate
for high sensitivity acquisition systems requiring more light in a
short time. Moreover, it appears that if the actual catalytic
efficiency of nanoKaz/NanoLuc is high, as it is in the 102-103
molS/s·molE range, the detection of emitted photons is
remarkably modest: only one RLU for more than 1800
decarboxylated molecules at best. Also of note is the fast
stochastic inactivation seen for some substrate in contrast with
other which may reflect the existence of two distinct
inactivation mechanisms which we are also trying to
investigate. In any case, in view of all this, we believe that
further luciferases mutagenesis and/or design of original
luciferin analogues could lead to even more improved
bioluminescence profiles.
Conclusions
In conclusion, the results of this work should herald more
researches focusing/based on bioluminescent reporting
systems using imidazo[1,2-a]pyrazine-3(7H)-one luciferins.
Indeed, we describe here a simple mean to prepare these rather
expensive luciferins in such amount that it should provide
scientists with many more opportunities to design and use such
reporting systems.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the Agence Nationale de la
Recherche (ANR), grant ANR-11-CRNT-0004, in the context of
the investment program ‘GLOBAL CARE, an association of the
Instituts Carnot ‘Pasteur-Maladies Infectieuses’, ‘Curie-Cancer’,
‘Voir et Entendre’, ‘Institut du Cerveau et de la Moelle Epinière’
and the ‘Consortium pour l’Accélération de l’Innovation et de
son Transfert dans le domaine du Lymphome’ (CALYM). E.P.C.
was supported by a fellowship from Global Care. This project
also benefited from the ValoExpress funding of the Institut
Pasteur. Prof. Christian Bréchot and Drs. Muriel Delepierre and
Daniel Larzul are acknowledged for their interest and support.
Finally, Victor Monnot‘s help is acknowledged for the syntheses
of some compounds.
Notes and references
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