New red-shifted coelenterazine analogues with an extended electronic conjugation

Article abstract of DOI:10.1016/j.tetlet.2012.07.041

A new promising approach to the development of red-shifted coelenterazine analogues was described. In order to alter the photochemical properties of native coelenterazine, we have designed and synthesized analogues bearing a new electron-rich structure. T

Full text of DOI:10.1016/j.tetlet.2012.07.041

Tetrahedron Letters 53 (2012) 5114–5118
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
New red-shifted coelenterazine analogues with an extended
electronic conjugation
Germano Giuliani a, , Paola Molinari b, Giulia Ferretti a, Andrea Cappelli a, Maurizio Anzini a,
⇑
Salvatore Vomero a, Tommaso Costa b
a Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, University of Siena, Via A. Moro 2, 53100 Siena, Italy
b Dipartimento di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new promising approach to the development of red-shifted coelenterazine analogues was described. In
order to alter the photochemical properties of native coelenterazine, we have designed and synthesized
analogues bearing a new electron-rich structure. The spectroscopic results obtained, in the presence of
the target enzyme (Renilla Luciferase), show a bathochromic emission shift of the entire class of new
derivatives. Among them, the 2-benzyl-8-(4-chlorophenylthio)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyra-
zin-3(7H)-one (8) shows an emission at 510 nm and uncommon slow kinetic decay.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 23 May 2012
Revised 6 July 2012
Accepted 10 July 2012
Available online 20 July 2012
Keywords:
Bioluminescence
Coelenterazine
Red-shift
2Y-System
Renilla Luciferase
The use of bioluminescent enzymes as gene reporters in cell
culture and small-animal imaging1,2 has become a useful techno-
logical tool for life science research and drug discovery. Most of
the bioluminescence systems investigated require a luciferase (en-
zyme), a luciferin (substrate), and molecular oxygen to produce
visible light.3 Enzymatic degradation of luciferin components pro-
duces intermediates bearing an electron in an excited state. More
in detail, the luminescence reaction is initiated by the binding of
O2 at the 2-position of the coelenterazine molecule, giving a perox-
ide. The peroxide then forms a four-membered ring ‘dioxetanone’,4
that promptly decomposes producing CO2 and the amide anion of
coelenteramide in its excited state. The excited state of the amide
anion of coelenteramide emits light when its energy level falls to
the ground state, resulting in the emission of blue light (kmax
450–470 nm).5 Clearly, the energy of the emitted photons depends
on the difference between the two energy states. For imaging
experiments in intact animals, photon emissions in the red to
near-infrared wavelengths (600–900 nm) are required, since the
tissue absorption of photons is reduced in this region of the spec-
trum.6 For this reason the firefly (beetle) luciferase that uses
D-
luciferin 1 (Fig. 1) as substrate (emission spectra in the range of
540–615 nm) is the preferred bioluminescent system in small-ani-
mal imaging applications. Unfortunately, the Mg2+- and ATP-
⇑
Corresponding author. Tel.: +39 0577 234359; fax: +39 0577 234333.
E-mail address: giuliani5@unisi.it (G. Giuliani).
Figure 1. General formula of the derivatives, where X is a not specified heteroatom.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.041

G. Giuliani et al. / Tetrahedron Letters 53 (2012) 5114–5118
5115
Figure 2. The spheres represent generic heteroatoms, in the 2Y-System in which
three of them are nitrogen atoms: N-1, N-4, and N-7 of the original imidazo[1,2-
a]pyrazin-3(7H)-one nucleus.
dependence of the reaction and the poor thermal stability of some
of these enzymes limit their versatility.
On the other hand, the reaction of imidazopyrazinone-type
luciferases that use coelenterazine 2 as the substrate [e.g. Renilla
(RLuc), Oplophorus, and Gaussia luciferases], only require is molec-
ular oxygen. Thus, they do not suffer from such problems, but the
emission range of these enzymes is in the blue region (450–
475 nm) of the visible spectrum. To overcome such a limitation,
the development of red-shifted and stable variants of RLuc
obtained via random mutagenesis7 used in combination with
red-shifted analogues of coelenterazine,8 has proven to be a prom-
ising approach.9 While it seems to be difficult to achieve additional
red-shifting by modifications of the enzyme at the current state of
knowledge, the synthesis of new coelenterazine analogues with
better bathochromic emission shifts may offer some room for
improvement. In fact, despite the large number of coelenterazine
analogues generated during the past forty years of research, only
few red-shifted substrates have been reported thus far. One of such
molecules, is v-coelenterazine, which displays a 35 nm bathochro-
mic shift and high quantum yield with RLuc, but not Oplophorus
Figure 3. Structures of the newly-synthesized coelenterazine analogues compared
to those of reference compounds 3 and 7.
also explored, in the attempt to further modulate the electron den-
sity of the 2Y-System. Similar substitutions were also made on h-
coelenterazine (7–8, Fig. 3), which has Rluc bioluminescent proper-
ties indistinguishable from the natural substrate.
luciferase.8,9 Additional coelenterazine analogues with
a
conjugated group at the C-8 position of the imidazopyrazinone ring
displayed large bathochromic shifts (50–110 nm) of chemilumi-
nescence in polar aprotic solvent, but their bioluminescence in
the Rluc reaction was negligibly low.10
The synthesis of the analogues 4,6,8 with sulfur heteroatom was
performed from commercially available 2-aminopyrazine (9) by
means of the procedure reported in Scheme 1 based on the Suzuki
coupling 12. Key pyrazine intermediates 11a–c13 were obtained by
reaction of bromoderivatives 10a,b14 with the appropriate thiol in
dry DMF or acetonitrile in the presence of sodium hydride as the
base.15 Demethylation of compound 11c was performed with pyr-
idine hydrochloride (PyÂHCl)12d at 190 °C to obtain the corre-
sponding hydroxyl derivative 11d.13
The final step of the convergent synthesis of the coelenterazine
analogues 4,6,816 was achieved by condensation of intermediates
11a,b,d, with diethoxy derivative17 in ethanol in the presence of
concentrated HCl.10
The introduction of the sulfur heteroatom in place of the meth-
ylene bridge in 3 leading to compound 4 produces a significant
(40 nm) bathochromic shift of the chemiluminescence spectrum
measured in neutral DMSO (Table 1). The p-chlorophenyl substitu-
ent did not change the chemiluminescence spectrum in the absence
of heteroatom (compound 518) nor did alter the red-shifting effect
of sulfur replacement (6). A similar red-shift (47 nm) of chemilumi-
nescent emission was observed in the h-coelenterazine derivative
(8, Table 1). This overall bathochromic shifting effect of C-8 hetero-
atom replacement was also maintained in the bioluminescent reac-
tion with Rluc. The bioluminescence spectra of 4 displayed a 30 nm
red-shift compared to 3 (Fig. 4). Likewise, both the minor shoulder
(kmax ꢀ387 nm) and the main peak (kmax 471 nm) in the biolumi-
nescent emission of 7 (believed to represent the neutral and the
On confronting 1 and 2, we note that in the D-luciferin molecule
N and S heteroatoms exist in a particular arrangement (see Fig. 2).
We suspect that this electron-rich configuration may play a funda-
mental role in the formation of the multiple oxyluciferin excited
states that result in a broad range of photon-emission colors.11
Thus, we hypothesize that building a similar heteroatomic config-
uration in the imidazopyrazinone scaffold (which we name
2Y-System, for heteroatoms stand symmetrically at the four tips
of a scaffold resembling an upside-down linked double Y) might
enhance the stability of lower-energy emitting species of
coelenterazine.
As a first step in this direction, we replaced with an S hetero-
atom the methylene group bonded to the C-8 of the imidazo[1,2-
a]pyrazin-3(7H)-one nucleus of 2, thus generating a series of hy-
brid compounds between the D-luciferin 1 and coelenterazine 2
(Fig. 1). By means of this simple structural modification, we as-
sumed to modulate the photoemission of the analogues, maintain-
ing the enzymatic binding properties of the native substrate. A
number of derivatives (3–6, Fig. 3) were made using bis-deoxycoel-
enterazine 3 as the reference, because this compound represents a
simpler coelenterazine analogue, which generates a lower-effi-
ciency blue-shifted luminescence (ꢀ400 nm) with Rluc. Together
with heteroatom interchange, S-phenyl halogen substitution was

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G. Giuliani et al. / Tetrahedron Letters 53 (2012) 5114–5118
Scheme 1. Reagents: (a) C6H5SH or 4-ClC6H4SH, NaH, dry CH3CN; (b) PyÁHCl; (c) 1,1-diethoxy-3-phenylacetone, concd HCl, H2O, EtOH.
Table 1
Chemiluminescence, bioluminescence, and relative quantum yields of coelenterazine analogues
Compd
Chemilum. kmax
Bioluminescencea
RQYb(biolum.)
RQYc(chemilum.)
1
2
kmax
kmax
3
4
5
6
7
8
457
497
456
499
460
507
390
419
382
469
471
509
—
—
432
—
387
432
1
1
3.6
1.3
0.2
688
23
1.83
1.80
0.92
0.96
0.42
a
b
c
Main peak (kmax1) and minor shoulder (kmax2) in the bioluminescent emission of analogues.
Bioluminescence quantum yield, photons emitted per molecule of luciferin reacted.
Chemiluminescence quantum yield, photons emitted per molecule of luciferin reacted.
Figure 5. Effect of the structural modifications on the corrected bioluminescence
emission spectra. Comparison between compounds 7 and 8.
Figure 4. Effect of the structural modifications on the corrected bioluminescence
emission spectra. Comparison between compounds 3 and 4.
amide anion emitting species, respectively) were red-shifted in
compound 8 (Fig. 5 and Table 1). However, while replacement of
only S in the bis-deoxycoelenterazine scaffold (3) resulted in
enhancement (ꢀfourfold) of bioluminescence efficiency, the
combined substitutions of both S and Cl caused a fivefold decrease.
Similarly S and Cl substitutions in h-coelenterazine (7) resulted in a
30-fold lower relative quantum yield (Table 1).
Interestingly, unlike chemiluminescence, the bioluminescence
of bis-deoxycoelenterazine derivatives was affected by chlorine
substitution of the S-linked phenyl ring. In fact, the red-shift

G. Giuliani et al. / Tetrahedron Letters 53 (2012) 5114–5118
5117
underlying mechanisms of bioluminescence in this class of
molecules.
Acknowledgments
We thank Professor P. Knochel and Dr. T. Bresser (Dep. Chemie
& Biochemie, LMU, Munchen, Germany) for their experimental
support and encouragement.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.07.
041.
References and notes
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Dev. Technol. 2008, 6, 787–793.
Figure 6. Bioluminescence spectrum of compound 5. The dotted lines indicate the
two emitting components resolved by the fitting procedure (see Section 4 of
Supplementary data).
2. (a) Watts, J. C.; Giles, K.; Grillo, S. K.; Lemus, A.; DeArmond, S. J.; Prusiner, S. B.
Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 2528–2533; (b) Patel, M. R.; Chang, Y. F.;
Chen, I. Y.; Bachmann, M. H.; Yan, X.; Contag, C. H.; Gambhir, S. S. Cancer Res.
2010, 70, 10141–10149.
3. Campbell, A. K. Chemiluminescence, principles and applications in biology and
medicine; Ellis Horwood Ltd: Chichester (England), 1988. pp. 189–218.
4. (a) Shimomura, O. Bioluminescence, chemical principles and methods; World
Scientific Publishing Co. Ltd: Singapore, 2006. pp. 159–176; (b) Liu, F.; Liu, Y.;
De Vico, L.; Lindh, R. J. Am. Chem. Soc. 2009, 131, 6181–6188; (c) Isobe, H.;
Yamanaka, S.; Okumura, M.; Yamaguchi, K. J. Phys. Chem. A 2009, 113, 15171–
15187.
5. Hori, K.; Wampler, J. E.; Cormier, M. J. Chem. Commun. 1973, 492–493.
6. Weissleder, R. Nat. Biotechnol. 2001, 19, 316–317.
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8. Inouye, S.; Shimomura, O. Biochem. Biophys. Res. Commun. 1997, 233, 349–353.
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Natl. Acad. Sci. U.S.A. 2011, 108, 12060–12065.
(ꢀ80 nm) with both Cl and S substitutions (6 vs 3, Table 1) was sig-
nificantly greater than that produced by only heteroatom replace-
ment (4). Moreover, Cl substitution in the absence of S (5)
determined a broadening of the bioluminescence spectrum, which
could be resolved as two components of higher and lower energy
(Fig. 6), suggesting that this compound generates two stable emit-
ters during the enzymatic reaction. Unfortunately, compound 6
also suffered a fivefold drop of bioluminescence efficiency com-
pared to 3; consequently the lower quality of spectral data did
not allow further analysis (Fig. S1).18
An additional interesting finding was only observed in the h-
coelenterazine derivative 8, and involved a sharp change of biolu-
minescence kinetics.18 Unlike 7, which shows the typical flash-like
kinetics of coelenterazines, with an immediate burst to peak emis-
sion rate followed by steady decay, the derivative 8 displayed a
more complex pattern. The initial burst was followed by a second
slower increase of emission rate, which lasted 5–7 s before giving
rise to the decay phase (Fig. S2). This peculiar difference in kinetics
was not observed in the chemiluminescent reaction (Fig. S3), which
suggests that the phenomenon is caused by different interactions of
the two molecules within the binding pocket of the enzyme.
The results of this study appear to support the idea that the
insertion of a C-8 bonded S atom, (thus achieving partial mimicry
10. Wu, C.; Nakamura, H.; Murai, A.; Shimomura, O. Tetrahedron Lett. 2001, 42,
2997–3000.
11. Naumov, P.; Ozawa, Y.; Ohkubo, K.; Fukuzumi, S. J. Am. Chem. Soc. 2009, 131,
11590–11605.
12. (a) Nakamura, H.; Aizawa, M.; Murai, A. Synlett 1996, 1015–1017; (b) DeBie, D.
A.; Ostrowicz, A.; Guertsen, G.; VanDerPlas, H. C. Tetrahedron 1988, 44, 2977–
2983; (c) Jones, K.; Keenan, M.; Hibbert, F. Synlett 1996, 509–510; (d)
Adamczyk, M.; Johnson, D. D.; Mattingly, P. G.; Pan, Y.; Reddy, R. E. Org. Prep.
Proced. Int. 2001, 33, 477–485.
13. General procedure for the synthesis of 11a–c: To
a cooled solution of the
appropriate thiophenol (1.0 equiv) in dry DMF, sodium hydride (1.2 equiv) was
added and the resulting mixture stirred at 5–10 °C for 15 min. Bromo
derivative 10a,b (1.0 equiv) was then added and the mixture was heated at
80 °C for 17–20 h. The reaction mixture was quenched with water and
extracted with EtOAc. The combined extracts were dried over anhydrous
Na2SO4 and concentrated under reduced pressure. Purification of the residue
by flash chromatography gave the target intermediates 11a–c (yield 74–80%).
Compound 11a: mp: 110–112 °C. 1H NMR (400 MHz, CDCl3) d: 8.37 (s, 1H, Ar-
H), 7.78 (d, J = 7.2, 2H, Ph-H), 7.53–7.50 (m, 2H, Ph-H), 7.41–7.29 (m, 6H, Ph-H),
5.02 (br s, 2H, NH2). 13C NMR (400 MHz, CDCl3) d: 151.8, 143.0, 137.8, 137.0,
136.6, 132.7, 130.7, 129.3, 128.8, 128.3, 125.5. MS (ESI) m/z: 280 [M+H+].
Compound 11b: mp: 157–158 °C. 1H NMR (400 MHz, CD3OD, 25 °C, TMS):
d = 8.29 (s, 1H; Ar-H), 7.68 (d, J(H;H) = 7.2 Hz, 2H, Ph-H), 7.51 (d,
J(H;H) = 8.4 Hz, 2H, Ph-H), 7.43 (d, J(H;H) = 8.8 Hz, 2H, Ph-H), 7.32 (t,
J(H,H) = 7.4 Hz, 2H, Ph-H), 7.24–7.27 (m, 1H, Ph-H). 13C NMR (400 MHz,
CD3OD, 25 °C, TMS): d = 152.7, 140.0, 137.4, 136.8, 136.7, 135.3, 133.7, 129.8,
129.7, 129.1, 128.2, 125.1. MS (ESI) m/z: 314 [M+H+]. Compound 11c:
Purification of the residue by flash chromatography using Al2O3, with
CH2Cl2ÁEtOAc (9:1 v/v) as the eluent, afforded compound 11c. mp: 154–
155 °C. 1H NMR (400 MHz, CDCl3, 25 °C, TMS): d = 8.28 (s, 1H; Ar-H), 7.68 (d,
J(H,H) = 8.8 Hz, 2H; Ph-H), 7.43 (d, J(H,H) = 8.8 Hz, 2H; Ph-H), 7.34 (d,
J(H,H) = 8.8 Hz, 2H; Ph-H), 6.91 (d, J(H,H) = 8.8 Hz, 2H; Ph-H), 4.90 (br s, 2H;
NH2), 3.82 (s, 3H; CH3). 13C NMR (400 MHz, CDCl3, 25 °C, TMS): d = 160.0,
151.0, 143.1, 136.9, 136.4, 134.4, 134.0, 129.3, 129.1, 126.7, 114.2, 55.4. MS
(ESI) m/z: 366 [M+Na+]. Compound 11d: To a Schlenk’s tube containing pyridine
hydrochloride (20 equiv) heated at 190 °C, compound 11c (1 equiv) was added.
The resulting mixture was stirred at 190 °C for 2 h under an argon atmosphere,
cooled at RT, and quenched with a saturated solution of NaHCO3. The resulting
mixture was extracted with EtOAc and the combined extracts were dried and
concentrated under reduced pressure. Purification of the residue by flash
chromatography using Al2O3, with CH2Cl2ÁEtOAc (1:1 v/v) as the eluent,
of the 2Y heteroatomic pattern in D-luciferin), can favor the emer-
gence of lower energy emitters in coelenteramide. This bathochro-
mic effect was evident in the presence of either the phenyl or the
phenol ring in C-6, indicating that the red shift can occur regardless
of whether the main emitter is the neutral or the amide-anion form
of the molecule. It also occurred in both chemiluminescence and
bioluminescence, suggesting that it likely results from an intrinsic
change in the electronic properties of the excited states of the mol-
ecule, independently of how they are produced. Fluorescence stud-
ies of coelenteramide analogues and model compounds suggest
that the lowest energy emitting species (530–550 nm) formed dur-
ing coelenterazine oxidation is the pyrazine-N(4) anion.19 We may
speculate that S replacement and the highly conductive 2Y pattern
that it generates may favor the formation of this anionic species.
However, further work is necessary to verify this possibility.
In conclusion, we describe here a new series of red-shifted
coelenterazine analogues and a novel approach to alter the photo-
chemical properties of the light-emitter intermediates. Further
exploitation of these findings may extend understanding on the

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G. Giuliani et al. / Tetrahedron Letters 53 (2012) 5114–5118
afforded compound 11d (yield 55%) as a yellow solid. mp: 133–134 °C. 1H NMR
0.1 mL), 25 °C, TMS): d = 139.9, 136.8, 136.2, 130.0, 129.7, 129.2, 129.1, 129.0,
128.9, 128.2, 126.8, 126.2, 108.5, 32.1. MS (ESI) m/z: 410 [M+H+]. Compound 6:
1H NMR (400 MHz, CDCl3, 25 °C, TMS): d = 8.13 (s, 1H; Ar-H), 7.53–7.50 (m, 2H;
Ph-H), 7.35–7.27 (m, 7H; Ph-H), 6.97 (d, J(H,H) = 6.8 Hz, 2H; Ph-H), 6.90–6.81
(m, 3H; Ph-H), 4.00 (s, 2H; CH2). 13C NMR (400 MHz, CDCl3, 25 °C, TMS):
d = 146.5, 142.1, 140.4, 138.6, 138.5, 136.8, 135.9, 135.8, 129.4, 128.9, 128.3,
128.2, 126.3, 125.8, 108.2, 31.5. MS (ESI, negative ions) m/z: 442 [M-H+].
Compound 8: mp: 166–167 °C. 1H NMR (400 MHz, CD3OD, 25 °C, TMS): d = 8.07
(s, 1H; Ar-H), 7.58 (d, J(H,H) = 8.4 Hz, 2H; Ph-H), 7.47–7.44 (m, 4H; Ph-H),
7.27–7.12 (m, 5H; Ph-H), 6.71 (d, J(H,H) = 8.8 Hz, 2H; Ph-H), 4.10 (s, 2H; CH2).
13C NMR (400 MHz, CD3OD, 25 °C, TMS): d = 159.4, 149.1, 147.3, 143.5, 140.6,
138.2, 136.7, 130.3, 129.5, 129.5, 128.4, 128.2, 127.9, 127.3, 116.4, 107.6, 97.6,
32.0. MS (ESI) m/z: 460 [M+H+].
(400 MHz, CD3OD, 25 °C, TMS): d = 8.18 (s, 1H; Ar-H), 7.52 (d, J(H,H) = 8.8 Hz,
2H; Ph-H), 7.47 (d, J(H,H) = 8.4 Hz, 2H; Ph-H), 7.39 (d, J(H,H) = 8.4 Hz, 2H; Ph-
H), 6.74 (d, J(H,H) = 8.8 Hz, 2H; Ph-H). 13C NMR (400 MHz, CD3OD, 25 °C, TMS):
d = 157.6, 151.2, 142.1, 137.3, 134.9, 134.6, 134.2, 129.4, 128.9, 127.9, 126.2,
115.1. MS (ESI, negative ions) m/z: 328 [MÀH+].
14. Sato, N. J. Heterocycl. Chem. 1978, 15, 665–670.
15. Shimazaki, M.; Hikita, M.; Hosoda, T.; Ohta, A. Heterocycles 1991, 32(5), 937–
947.
16. General procedure for the synthesis of 4,6,8: A mixture of the appropriate
pyrazine derivative (11a,b,d 1 equiv), 1,1-diethoxy derivative 12 (1.4 equiv),
36% aqueous HCl (13 equiv) and ethanol (10 mL) was refluxed for 5–8 h. After
cooling to RT, the reaction mixture was concentrated under reduced pressure
and the residue obtained was purified by flash chromatography with
CH2Cl2ÁMeOH (95:5 v/v) as the eluent to obtain the corresponding
coelenterazine derivative (4,6,8, yield 25–45%). Note: final compounds
should be protected from the exposure to air, light and stored at À18 °C.
Compound 4: mp: 115–117 °C. 1H NMR (400 MHz, CD2Cl2, 25 °C, TMS): d = 8.12
(s, 1H; Ar-H), 7.65–7.59 (m, 4H; Ph-H), 7.49–7.45 (m, 2H; Ph-H), 7.32–7.11 (m,
9H; Ph-H), 4.10 (s, 2H; CH2). 13C NMR (400 MHz, CD2Cl2/CD3OD (0.4 mL/
17. Adamczyk, M.; Johnson, D. D.; Mattingly, P. G.; Pan, Y.; Reddy, R. E. Synth.
Commun. 2002, 32, 3199–3205.
18. Synthetic scheme and procedure of compound 5, material and methods for
luminescent measurement are reported in detail in the Supplementary data.
For bioluminescent spectra of compound 6, kinetic plots of 7 and 8 see section
7 of the Supplementary data.
19. Shimomura, O.; Teranishi, K. Luminescence 2000, 15, 51–58.