Toward bioluminescence in the near-infrared region: Tuning the emission wavelength of firefly luciferin analogues by allyl substitution

Article abstract of DOI:10.1016/j.tetlet.2018.01.078

The synthesis and bioluminescence of allyl-substituted luciferin derivatives as substrates for firefly luciferase are reported. The allylation of luciferins induced bathochromic shift (15–40 nm) of the bioluminescence emission. Upon combination with other chemical modifications for bioluminescence wavelength tuning, novel red emitting luciferin analogues were obtained with emission maxima at 685 and 690 nm.

Full text of DOI:10.1016/j.tetlet.2018.01.078

Accepted Manuscript
Toward Bioluminescence in the Near-Infrared Region: Tuning the Emission
Wavelength of Firefly Luciferin Analogues by Allyl Substitution
Nobuo Kitada, Tsuyoshi Saitoh, Yuma Ikeda, Satoshi Iwano, Rika Obata,
Haruki Niwa, Takashi Hirano, Atsushi Miyawaki, Koji Suzuki, Shigeru
Nishiyama, Shojiro A. Maki
PII:
S0040-4039(18)30123-0
https://doi.org/10.1016/j.tetlet.2018.01.078
TETL 49665
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 December 2017
24 January 2018
26 January 2018
Please cite this article as: Kitada, N., Saitoh, T., Ikeda, Y., Iwano, S., Obata, R., Niwa, H., Hirano, T., Miyawaki,
A., Suzuki, K., Nishiyama, S., Maki, S.A., Toward Bioluminescence in the Near-Infrared Region: Tuning the
Emission Wavelength of Firefly Luciferin Analogues by Allyl Substitution, Tetrahedron Letters (2018), doi: https://
doi.org/10.1016/j.tetlet.2018.01.078
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Tetrahedron Letters
Toward Bioluminescence in the Near-Infrared Region: Tuning the Emission
Wavelength of Firefly Luciferin Analogues by Allyl Substitution
Nobuo Kitada,a Tsuyoshi Saitoh,b, c* Yuma Ikeda,d Satoshi Iwano,e Rika Obata,f Haruki Niwa,a Takashi
Hirano,a Atsushi Miyawaki,e Koji Suzuki,d Shigeru Nishiyama,C and Shojiro A. Makia*
a Department of Engineering Science, Graduate School of Informatics and Engineering, University of Electro-Communications, Chofu, Tokyo, 182-8585
b
International Institute for Integrative Sleep Medicine, University of Tsukuba, Tennodai 1-1-1, Tsukuba-shi, Ibaraki, 305-8577
c Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama, Kanagawa, 223-8522
d Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama, Kanagawa, 223-8522
e Laboratory for Cell Function Dynamics Brain Science Institute, RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198
f Research and Education Center for Natural Sciences, Keio University, Hiyoshi 4-1-1 Kohoku-ku, Yokohama 223-8521
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The synthesis and bioluminescence of allyl-substituted luciferin derivatives as substrates for
firefly luciferase are reported. The allylation of luciferins induced bathochromic shift (15–40
nm) of the bioluminescence emission. Upon combination with other chemical modifications for
bioluminescence wavelength tuning, novel red emitting luciferin analogs were obtained with
emission maxima at 685 and 690 nm.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Luciferin derivatives
Bioluminescence
Near infrared
Firefly

2
Luminescent (e.g. fluorescent and bioluminescent) systems that emit light in the red or near-infrared (NIR) region have recently
been reported as powerful chemical probes for the observation of biological phenomena and other applications.1–4 In particular, the
demand for NIR-luminescent systems for non-invasive and operationally simple diagnosis techniques, such as in vivo imaging, has
grown drastically.5 The optical window (650–950 nm) is expected to allow sensitive detection during in vivo imaging by avoiding
unexpected absorption of visible and IR light by living specimen. While various NIR-fluorescent systems have been developed,
including cyanines6,7 or boron-dipyrromethenes (BODIPYs),8 only few NIR-bioluminescent systems have been reported.9–16 In the
luciferin bioluminescence reaction, photons are emitted from the excited state of oxyluciferin (2), which is a metabolic product of
the firefly luciferin (1a) after adenylation by firefly luciferase, followed by a reaction with molecular oxygen (Figure 1).17 The
excited state of 2 emits only at ca. 560 nm in the presence of natural luciferase. During the course of our studies on the development
of luciferin analogues,10,11,16 we discovered that the replacement of benzothiazole with other aromatic rings causes a dramatic shift
of the emission wavelength. We have described a method to tune the emission wavelength of luciferins (Figure 1), in which the
extension of the π-conjugation between the thiazoline moiety and the aromatic ring affords a large bathochromic shift (ca. 100 nm),
while substitution of the hydroxide moiety with dimethylamine provides a smaller bathochromic shift (ca. 30
Figure 2. Structure of luciferins 1-6.
Figure 1. Bioluminescence reaction mechanism and structure–
wavelength relationship for luciferins.
nm).10 This approach has been applied to other luciferin derivatives, such as naphthyl luciferin18 (NapLuc, 3a; n = 0) and
dimethylanilyl luciferin (DmaLuc, n = 0) to obtain the red-emitting vinyl analogues NapVLuc (4a), DmaVLuc (5a), and
DmaDVLuc (6a) (Figure 2). Especially 6a exhibits promising bioluminescence properties in the NIR region (em = 680 nm) that
could potentially be exploited for the in vivo imaging of deep tissue.19,20 However, the further extension of the π-conjugation of
luciferin analogues renders the compound instable, resulting in a dramatic suppression of the luminescence activity. Another type of
NIR luciferins has been developed based on the modification of D-aminoluciferin. Cyclic alkylaminoluciferins (CycLucs)14 and N-
cycloalkylaminoluciferins such as cybLuc15 exhibit red (600–650 nm) bioluminescence and these luciferins can be used for in vivo
bioimaging of deep tissue such as brain tissue.15,21 Modifications of D-luciferin (1a) by halogenation,22–24 alkylation,25 and
alkynylation26 at the C-4’, -5’, and -7’ positions have also been reported to produce intriguing bioluminescence features. Recently,
we have reported the synthesis of C-7’ ally luciferin (1b) and its interesting bio-orthogonal luminescence properties.27 The C-7’
allylation of 1a induced a bathochromic shift (40 nm) of the emission wavelength, implying a weaker enzymatic recognition of the
benzothiazole ring and the formation of an excited state with lower energy. However, the detailed effects of such an allyl
substitution on the bioluminescence emission of other known luciferase substrates remain to be elucidated. Based on these
observations, we herein report the application of said allylation method for the further tuning of the bioluminescence wavelength of
known luciferins.

3
The effect of allyl substitution on the firefly bioluminescence of luciferin derivatives (3b, 4b, 5b, and 6b) was investigated
(Figure 2). The synthesis of 3b started with the O-allylation of 6-cyano-2-naphthol (7) to afford an allyl ether, followed by a Claisen
rearrangement, which selectively proceeded at the C-7’ position to afford 8 in high yield. A cyclocondensation with D-cysteine
Scheme 1. Syntehsis allyl-substituted luciferins (3b, 4b, 5b, 6b)
ultimately converted 8 into 5’-allyl NapLuc (3b) (Scheme 1A). To access 5’-allyl NapVLuc (4b), a twofold O-allylation of
commercially available 6-hydroxy-2-naphthoic acid (9) was carried out, followed by a Claisen rearrangement to afford 5’-allyl
naphthalene 10. After protection of the hydroxyl group with TBS, the allyl ester was reduced with DIBALH and subsequently
oxidized into aldehyde 11 (Scheme 1B). A Wittig reaction then furnished a vinyl ester, which yielded carboxylic acid 12 upon
hydrolysis with NaOH aq. Subsequently, 12 was condensed with H-D-Cys(Trt)-OMe, followed by the formation of a thiazoline ring
with Tf2O and OPPh328 to furnish 13. The desired luciferin 4b was ultimately isolated following the hydrolysis of the methyl ester
moiety. Similarly, the synthesis of 3’-allyl DmaLuc (5b) and DmaVLuc (6b) started with the bromination of commercially available
4-dimethylaminobenzaldehyde (14), followed by a Pd-catalyzed Stille coupling with allyltributyltin to afford allyl arene 15 (Scheme
1C). A Wittig or Horner–Wadsworth–Emmons reaction of the aldehyde with phosphonic acid diester 16 afforded the corresponding
vinyl and dienyl esters, which yielded the carboxylic acids 17 and 18 upon hydrolysis with NaOH aq. The desired luciferins 5b and
6b were isolated in the same manner as 4b.

4
The bioluminescence (BL) spectra of the synthetic luciferins with recombinant firefly luciferase (Ppy; Photinus pyralis) are

5
Figure 3. Normalized bioluminescence emission spectra for 3a (λmax = 555 nm), 3b ((λmax = 570 nm), 4a (λmax = 655 nm), 4b
(λmax = 690 nm), 5a (λmax = 565 nm), 5b (λmax = 575 nm), 6a (λmax = 670 nm), and 6b (λmax = 685 nm).
Table 1. Luminescence wavelength and kinetic properties of
the synthetic luciferins 1-6
BL
CL
e
Comp.
rel. Vmax
Kmf [µM]
λ
λ
max
max
[nm]a
[nm]b
570
585c
100
4.1
160 ± 58g
370 ± 50
58 ± 13
1a
1b
3a
3b
4a
4b
5a
5b
6a
6b
610
555
570
655
690
565
575
670
685
580c
515c
520c
605c
610c
580d
580d
665d
660d
0.096
0.12
0.10
0.13
6.3
28 ± 5.6
3.2 ± 0.88
3.3 ± 0.54
4.4 ± 2.0
71 ± 27
0.10
1.9
2.0 ± 0.3
17 ± 4.1
0.12
b
a
Bioluminescence emission maxima; Chemiluminescence emission
c
maxima; Chemiluminescence was measured using 1.25 mM luciferin
methyl ester and 125 mM t-BuOK in DMSO; d Chemiluminescence was
e,f
measured using 1 mM luciferin, T3P, and Et3N in DMF;29 The kinetic
constants are apparent values, determined by measurement of the initial
rates of light emission from 0.02 µM to 200 µM luciferin, 2 µg Ppy and
80 µM ATP-Mg.30,31 The rel. Vmax values were shown by comparison
g
with the value for 1a.
The Km value strongly depends on the
measurement conditions. The value of 1a obtained in this study is
essentially consistent with previously reported data that were measured
under identical conditions.18,31
shown in Figure 3. The allylated NapLuc 3b displays a bathochromic shift (15 nm) of the emission maximum compared to that of
3a (Table 1). On the other hand, allylated NapVLuc 4b exhibits a larger bathochromic shift (35 nm) of its peak maximum relative to
that of 4a, with an emission wavelength similar to that of 1b. Regarding the effect of allyl substitution on the DmaLuc derivatives,
5b (10 nm) and 6b (15 nm) display bathochromically shifted peak maxima compared to the non-allylated compounds. We then
measured the chemiluminescence (CL) spectra, which reflect the energy level of the excited states without any effect of the enzyme,
in order to evaluate the effect of allyl substitution. However, the observed chemiluminescence maxima of allylated luciferins (1–6b)
are not significantly changed relative to those of the original luciferins (1–6a), indicating that allyl substitution on the aromatic rings
of luciferin does not significantly affect the energy levels of the oxyluciferins in excited singlet state. Because relative Vmax (rel.
Vmax) values were determined by using the data of emitted photons with a constant concentration of the luciferase, a rel. Vmax value is
proportional to the product of the rate constant of the overall reaction (kcat) and the bioluminescence quantum yield (Φbl). The rel.
Vmax values of 3a and 3b are similar to each other, and 4a and 4b also have similar rel. Vmax values, indicating that the allyl
modification to the hydroxyl-substituted analogues has an insignificant effect on the kcat and Φbl properties. Interestingly, the rel.

6
Vmax values of 5b and 6b are one order of magnitude less than those of 5a and 6a, respectively. This result indicates that the allyl
modification to the dimethylamino-substituted analogues affects an interaction between the substrate and the luciferase, resulting in
a decrease of the reaction rate and/or the efficiency of photon production. The bathochromic shifts observed in 5b and 6b are
probably related to the Km values, which are higher than those of 5a and 6a, implying a weaker enzymatic recognition similar to
1b.27 However, even though NapLuc derivatives also showed bathochromic shifts, the kinetic properties indicated equally strong
enzymatic recognition. This result probably implies a different binding mode for NapLuc derivatives with luciferase, which
stabilized the conformation of lower energy state of their metabolites. The in vivo bioimaging with NIR substrates 4b and 6b was
investigated in CAG-ffLuc-cp156 transgenic mice33 (Fig. 4A). Low but detectable bioluminescence was observed for each substrate.
Interestingly, the overall photon output of 4b and 6b was 2.3 and 3.4%, respectively, compared to that of 1a in spite of the lower
Vmax values (Fig. 4B). 6a also exhibited brighter in vivo than in vitro bioluminescence, due to the higher membrane permeability,
lower saturation concentration, and NIR bioluminescence.19 The photon output of 4b and 6b should accordingly also be enhanced
due to these bioluminescence and pharmacokinetic properties.
In conclusion, we have synthesized a series of allylated luciferins as prospective bioluminescence probes. Allylic substitution on
the aromatic moieties induces small but detectable bathochromic shifts (15–40 nm) of the emission wavelength. This approach can
be combined with other known methods for the tuning of the bioluminescence wavelength. The luciferin analogues 4b and 6b
exhibit NIR emission at 690 and 685 nm, respectively. To our knowledge, 4b is thus the substrate with the longest emission
wavelength for native firefly luciferase. Recently, a significant improvement of luminous efficacy of synthetic luciferins with
mutant luciferases has been reported.14,25,34 We have also reported the enzyme specificity of 7’-allyl luciferin (1b), i.e., the
combination with 1b and Eluc, a click beetle luciferase, improved the luminous efficacy.27 Although the NIR luciferins reported
here exhibit much lower emission intensity, it should be possible to improve the luminous efficacy with mutant luciferase(s). Most
importantly, the modification strategy for known luciferins should provide access to the next generation of NIR bioluminescence
tools.
Acknowledgments
This work was supported by the Grant-in-Aid for Scientific Research on Innovative Areas “Resonance Bio” (15H05948; S.A.M.)
and a JSPS research fellowship for young scientists (N.K.) from the Ministry of Education, Culture, Sports, Science and
Technology, Adaptable & Seamless Technology Transfer Program through Target-driven R&D (A-Step) (AS2614119N; S.A.M.)
from the Japan Science and Technology Agency, and a Kurata grant (1207; T.S.) from The Kurata Memorial Hitachi Science and
Technology Foundation.
Supplementary data
Supplementary data (characterization, bio- and chemiluminescence assays and bioluminescence imaging) associated with this
article can be found in the online version at XXX.
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1a (exposure 1 s)
6a (exposure 1 s)
(A)
Iv injection
Gain: 10
Substrate:
0.1 µmol/g BW
5,000
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Highlights


Allyl-substituted luciferin derivatives were designed and synthesized.
Bioluminescence properties of allyl luciferin derivatives were evaluated.

8


The allylation of luciferins induced bathochromic shift of their bioluminescence.
The longest near-infrared bioluminescence emission wavelength was achieved.
Graphical abstract
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Toward Bioluminescence in the Near-
Infrared Region: Tuning the Emission
Wavelength of Firefly Luciferin Analogues
by Allyl Substitution
Leave this area blank for abstract info.
Nobuo Kitada, Tsuyoshi Saitoh, Yuma Ikeda, Satoshi Iwano, Rika Obata, Haruki Niwa, Takashi Hirano,
Atsushi Miyawaki, Koji Suzuki, Shigeru Nishiyama, and Shojiro A. Maki
CO 2
H
Allyl substitution
N
π-extention
CO 2
H
S
CO 2
H
N
N
HO
S
S
HO
HO
1989s
2013s
2017s
450
500
550
600
Wa velength(nm)
650
700
750