Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo bioluminescence imaging

Article abstract of DOI:10.1016/j.tet.2017.11.051

The dimethylamino group of AkaLumine ((4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in the red region (656–667 nm). In particular, a bioluminescence reaction of 1-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher quantum yield than that with AkaLumine, giving an improved bioluminescence intensity. The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body luciferase-expressing mice among the analogues, indicating that a quantum yield improvement of a luciferin analogue is effective to increase bioluminescence imaging intensity.

Full text of DOI:10.1016/j.tet.2017.11.051

Accepted Manuscript
Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo
bioluminescence imaging
Masahiro Kiyama, Satoshi Iwano, Satoshi Otsuka, Shijia W. Lu, Rika Obata, Atsushi
Miyawaki, Takashi Hirano, Shojiro A. Maki
PII:
S0040-4020(17)31210-3
10.1016/j.tet.2017.11.051
TET 29131
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 6 October 2017
Revised Date: 14 November 2017
Accepted Date: 17 November 2017
Please cite this article as: Kiyama M, Iwano S, Otsuka S, Lu SW, Obata R, Miyawaki A, Hirano
T, Maki SA, Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo
bioluminescence imaging, Tetrahedron (2017), doi: 10.1016/j.tet.2017.11.051.
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Red-light-emitting
(λbl = ca. 655 - 670 nm)
ACCEPTED MANUSCRIPT
e2
Graphical abstract

ACCEPTED MANUSCRIPT
Quantum Yield Improvement of Red-light-emitting Firefly Luciferin
Analogues for in vivo Bioluminescence Imaging.
a
b
a
a
c
Masahiro Kiyama , Satoshi Iwano , Satoshi Otsuka , Shijia W. Lu , Rika Obata ,
b
a
a,
Atsushi Miyawaki , Takashi Hirano , Shojiro A. Maki *
a
Department of Engineering Science, Graduate School of Informatics and Engineering,
The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan.
b
Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Hirosawa
2
-1, Wako, Saitama 351-0198, Japan.
c
Research and Education Center for Natural Sciences, Keio University, Hiyoshi 4-1-1,
Kohoku-ku, Yokohama, Kanagawa 223-8521, Japan.
*
E-mail: s-maki@uec.ac.jp
Keywords
Firefly luciferin; Red-light-emitting; Enzyme kinetics; Quantum yield; Bioluminescence
imaging.
1

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Abstract
The
(4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazol
dimethylamino
group
of
AkaLumine
(
ecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic
amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give
AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in
the red region (656 to 667 nm). In particular, a bioluminescence reaction of
1
-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher
quantum yield than that with AkaLumine, giving an improved bioluminescence intensity.
The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body
luciferase-expressing mice among the analogues, indicating that a quantum yield
improvement of a luciferin analogue is effective to increase bioluminescence imaging
intensity.
1
. Introduction
Noninvasive in vivo bioluminescence imaging (BLI) is now widely used for
monitoring molecular and cellular events in small animals as a sensitive, rapid and
1
inexpensive method. The combination of luciferase of the North American firefly
2

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Photinus pyralis (Ppy) and its wild type substrate D-luciferin (LH ) is most commonly
2
used for BLI. Firefly bioluminescence (BL) is based on a luciferin-luciferase (L-L)
reaction involving the enzymatic action of luciferase reacting with luciferin as a
2
substrate. That is, the luciferase catalyzes the reaction of LH with ATP and oxygen in
2
2
+
the presence of Mg to give the light emitter, oxyluciferin (OLH), in the excited singlet
state (Figure 1A). The L-L reaction of LH with Ppy luciferase generates yellow green
2
light (BL maximum wavelength [λ ] = 560 nm) with a high quantum yield, which was
bl
3
0
.48 in a buffer at pH 8.0. Because BL is based on a chemical reaction, there is no need
to use an excitation light source like a fluorescence assay would. Thus, BLI provides
image data with lower signal-to-noise ratio than fluorescence imaging.
The yellow green light generated by the L-L reaction of LH with Ppy
2
luciferase is readily absorbed by mammalian tissues containing hemoglobin and
4
oxyhemoglobin, resulting in weakly transmitted light in BLI assays. To improve tissue
penetration of emitted light for BLI, it is necessary to use L-L reactions generating light
in the red to near-infrared (NIR) radiation (λ = 650–900 nm) in the optical window for
bioimaging. For this purpose, we and other groups have been developing luciferin
5
analogues to emit light in the red to NIR region.
Because of a high selective substrate specificity of luciferase, luciferin
3

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analogues were designed based on the LH structure. As a pioneering work, it was
2
found that aminoluciferin having the amino group instead of the hydroxyl group at the
C6’ position in LH shows an efficient BL with the maximum wavelength at ca. 600 nm
2
6
with Ppy luciferase (Figure 1B). As a method to modify the aminoluciferin structure,
aminoluciferin analogues conjugating a fluorescent dye at the amino group were
systematically prepared. BL of the analogues conjugating BODIPY and Cy 7 (Figure
1
B), for instance, showed the λbl values at 679 and 772 nm, respectively, by
7
intramolecular Förster resonance energy transfer. As the other method, the amino group
of aminoluciferin was converted to the fused azacycle, to give Cycluc series analogues
8
(
λ ≤ 648 nm). Cycluc1 with the fused pyrrolidine (Figure 1B), for instance, showed a
bl
9
high performance in BLI. As a modified aminoluciferin, an analogue with a selenium
atom in the thiazoline ring was also prepared, showing BL with the λ value at 600 nm
bl
1
0
(
Figure 1B).
In contrast to these methodologies, we have designed luciferin analogues by
modification of the π-electronic structure of the benzothiazole moiety to the
11
2
-phenylethenyl and 4-phenylbutadieneyl structures. Because the hydroxyl and amino
groups of LH and aminoluciferin, respectively, work as electron-donating substituents
2
to increase BL activities, the hydroxyl and dimethylamino groups were introduced on
4

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the phenyl group of these analogues. Among the π-modified analogues, the
4
-(4-dimethylaminophenyl)butadienyl
analogue
1a
((4S)-2-[(1E,3E)-4-[4-(dimethylamino)
phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid; Figure 1C) showed a
moderate BL activity with the longest λ value (675 nm) using Ppy luciferase. This
bl
analogue and its HCl salt have been commercialized with names of AkaLumine (Wako
Pure Chemical Industries, Ltd., Osaka, Japan) and Tokeoni (Sigma-Aldrich Co. LLC.,
St. Louis, MO, US), respectively. Because of the red-shifted spectroscopic property of
1
a, luminescence intensities of BLI for mammals with Tokeoni were significantly
1
2
stronger than those with LH and Cycluc1. In a similar way to modify the π-electronic
2
structure, luciferin analogues with the ethylene and butadiene moieties inserted between
the benzothiazole and thiazoline moieties of LH were prepared. The ethylene-inserted
2
analogue showed BL using mutant Ppy luciferase with the λ value at 706 nm (670 nm
bl
1
3
for wild type Ppy; iLH ; Figure 1B).
2
As a red-light-emitting luciferin analogue, AkaLumine (1a) is an attractive
compound as a prototype to design new luciferin analogues. The molecular shape of 1a
is linear and the butadienyl moiety is flexible enough to fit the molecular space of the
luciferin active site for a desirable BL activity. To improve the BL properties of 1a, we
5

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planned to fine-tune the molecular structure of 1a with keeping the backbone structure.
As a method to fine-tune the luciferin structure, we recently reported the BL properties
1
4
of aminoluciferin analogues having a cyclic amino group at C6’ (Figure 1C). The
cyclic amino groups have similar electron donating properties to dimethylamino group,
while the molecular volumes and hydrophobicities of the cyclic amino groups are
greater than those of dimethylamino group. Thus, the molecular size and hydrophobicity
of luciferin analogues are successfully tuned by changing the ring size of the cyclic
amino group. In fact, the analogues with the cyclic amino groups showed higher BL
quantum yields (Φ ) than 6’-dimethylaminoluciferin, with the λ values around 625 nm.
bl
bl
In this study, we applied the fine-tuning methodology to the AkaLumine system. We
prepared here AkaLumine analogues 1b–e having a cyclic amino group (Figure 1C) and
investigated their BL properties. Furthermore, BLI properties of the analogues were also
investigated and were evaluated with comparison to the fundamental BL properties.
6

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Figure 1. Structures of (A) firefly luciferin (LH ) and oxyluciferin (OLH), (B)
2
previous luciferin analogues, and (C) cyclic aminoluciferin and AkaLumine (1a) and
new luciferin analogs (1b–e).
2
. Results and Discussion
2
.1. Synthesis of AkaLumine analogues with a cyclic amino group
AkaLumine analogues (1b–e) with 1-pyrrolidinyl, 1-piperidino, 1-azepanyl and
7

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morpholino groups were synthesized from the corresponding benzaldehydes 2b–e by
11
the procedure shown in Figure 2 (see detailed procedure in ESI section 2).
4
-(1-Azepanyl)benzualdehyde 2d was prepared by ipso-substitution of
-fluorobenzaldehyde with hexamethyleneimine. Benzaldehydes 2b–e were
4
transformed into diene ethyl esters 3b–e by Horner-Wadsworth-Emmons reactions with
-phosphonocrotonate. Esters 3b–e were hydrolyzed followed by condensations with
4
S-trityl-
D
-cysteine methyl ester ( -Cys(Trt)-OMe), to give amides 4b–e. Thiazoline
D
rings of 5b–e were formed by cyclizations of the amide moieties with
1
5
trifluoromethanesulfonic anhydride (Tf O), leading to thiazoline esters 5b–e. Finally,
2
acid hydrolysis of 5b–e gave analogues 1b–e.
Figure 2. Synthesis of AkaLumine analogues 1b–e. Reaction conditions: (a)
Hexamethyleneimine, K CO , DMF; (b) Triethyl 4-phosphonocrotonate, NaH, THF; (c)
2
3
8

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i) NaOH aq., iPrOH; ii)
D
-Cys(Trt)-OMe, EDC, DMF; (d) Tf O, CH Cl ; (e) HCl aq.
2
2
2
2
.2. Bioluminescence properties of AkaLumine 1a and its analogues 1b–e.
L-L reactions of 1b–e using Ppy luciferase in pH 8.0 buffer were investigated
together with that of 1a. BL emission spectra for 1a–e are shown in Figure 3, and BL
properties are summarized in Table 1. 1-Piperidino analogue 1c showed single band
spectrum similar to that of 1a, and the λ values of 1a (668 nm) and 1c (667 nm) are
bl
similar to each other. BL spectra of 1b, 1d and 1e show main emission bands around
6
70 nm with minor emission bands around 560 nm. The λ values of the main bands of
bl
1
b, 1d and 1e were observed at 667, 656 and 665 nm, respectively. Interestingly, the
main emission band of 1d is 12 nm blue shifted compared to that of 1a. The minor
emission bands of 1b, 1d and 1e were observed in the wavelength region similar to that
of LH . In particular, 1d showed the minor emission band significantly.
2
9

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Figure 3. BL spectra of 1a–e.
Table 1. BL properties of 1a–e with Ppy luciferase and fluorescence properties of 1a–e
in methanol.
a
b
c
d, e
c
f
Compound λ / nm
Φbl
Km / ꢀM
rel. Vmax
rel. kcat
λfl / nm (Φf
)
bl
1
1
a
668
0.0050 0.14
0.020 0.22
1.0
1.0
537 (0.016)
545 (0.017)
b
(565), 667
667
2.2
0.54
0.37
2.8
1
1
1
c
d
e
0.0064 0.53
0.0062 0.71
0.0043 2.3
0.47
3.5
539 (0.019)
549 (0.020)
534 (0.016)
567, 656
(565), 665
0.81
0.96
a
b
BL maximum wavelength. BL quantum yield relatively determined with the value of LH (Ref. 3).
2
c
d
The errors are within 10%. The standard errors are within 10%. Relative Vmax and kcat value was
e
calculated by comparison with the value for 1a at pH 8.0. The standard errors are within 8%.
f
Relative Kcat value was calculated by dividing rel. Vmax by BL quantum yield. Fluorescence
maximum wavelength (λfl) and quantum yield (Φ ) in parenthesis.
f
To evaluate of the λ values of 1a–e, spectroscopic properties of 1a–e in
bl
methanol and a pH 8.0 buffer were investigated, and the fluorescence and UV/vis
absorption data are summarized in Tables S1 and S2. Fluorescence emission maximum
10

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wavelengths (λ ) of 1a–e in methanol were observed at 537, 545, 539, 549 and 534 nm,
fl
respectively (Table 1). Because the λ values of a luciferin analogue and the
fl
1
6
corresponding oxyluciferin analogue are correlated with each other, the λ values of
fl
1
a–e are useful to predict the λ values of the corresponding oxyluciferin analogues
fl
oxy-1a–e, which are the light emitters of the L-L reactions of 1a–e. That is, the λfl
values of 1a–e strongly correlate with their λ values. The λ values of 1a–e indicate
bl
fl
that 1-pyrrolidinyl and 1-azepanyl groups have electron donating properties slightly
stronger than dimethylamino group, resulting in a small red shift of the λ values.
fl
1
-Piperidino group has similar electron donating properties to dimethylamino group.
The electron donating property of morpholino group is slightly weaker than that of
dimethylamino group, leading to a small blue shift of the λ value. The substituent
fl
dependent variation of the λ values of 1a–e is similar to that of cyclic aminoluciferin
fl
1
4
analogues. In contrast, the λ values of the main emission bands of 1b, 1c and 1e are
bl
similar to that of 1a, while 1d showed a small blue shift of the λ value of the main
bl
emission band, indicating that the excited singlet states of oxy-1a–e generated by the
L-L reactions do not reflect the fluorescence properties in a homogeneous solution. That
is, the λ value of 1a–e are modulated by the active site of Ppy luciferase, in which
bl
steric and hydrophobic properties of the cyclic amino groups affected the emission
11

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properties of the excited singlet states of oxy-1a–e.
BL quantum yields (Φ ) of 1a–e were measured relatively using the reported
bl
3
value of LH with Ppy luciferase as a standard. The Φ values of 1a–e were estimated
2
bl
as 0.0050, 0.020, 0.0064, 0.0062 and 0.0043, respectively, indicating that the Φ value
bl
of 1c–e are similar to that of 1a, and 1-pyrrolidinyl analogue 1b showed the largest
value among them. While fluorescence quantum yields (Φ ) of 1a–e in methanol were
fl
almost identical (ca. 0.02), the high Φ value of 1b is possibly caused by a particular
bl
improvement of Φ of oxy-1b in the luciferase active site. As another cause, there is a
fl
possibility of a special increase in the chemiexcitation efficiency for the L-L reaction of
1
b. The molecular size and hydrophobicity of the 1-pyrrolidinyl group in 1b play an
important role in increasing the Φ value due to a better fit to the luciferase active site
bl
compared to those of the dimethylamino group in 1a.
Kinetic constants (K and Vmax) of 1a–e were estimated by the reported
m
1
7
methods (Table 1). The K values of 1b–e (0.22–2.3 µM) were greater than that of 1a
m
(0.14 µM), indicating that a bulkiness of the cyclic amino groups promotes ejection of
the analogues from the luciferase active site. In particular, morpholino analogue 1e
showed the largest K value among them, indicating that a weakened hydrophobic
m
property of the morpholino group decreases the affinity of 1e to the luciferase active
12

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site.
Relative Vmax (rel. Vmax) and relative kcat (rel. k ) were determined as relative
cat
values compared with those of 1a (Table 1). The rel. Vmax values of 1b–e are larger than
that of 1a. In particular, 1b and 1d showed 2.2 and 3.5 times larger values than 1a,
1
7
respectively. The rel. kcat value was calculated by dividing rel. Vmax by Φbl.
Interestingly, only 1-azepanyl analogue 1d showed larger rel. kcat value than 1a,
suggesting that the flexible property of the 1-azepanyl ring of 1d causes an inclusion of
1
d in the luciferase active site to accelerate the enzymatic reaction as observed in
1
4
6
’-(1-azepanyl)luciferin from previous research.
2
.3. DFT Calculations of 1a–e and the corresponding oxyluciferins oxy-1a–e.
To evaluate the λ values of 1a–e based on the electronic properties, we carried
bl
out DFT and TD-DFT calculations of 1a–e and the corresponding oxyluciferins oxy-1a–
e with the B3LYP/6-31+G (d) method. The results are summarized in Table 2. The
optimized structures of 1a–e with the lowest heats of formation has the conformation of
the single bond between the thiazoline ring and the diene moiety shown in Figure 4.
1
8
This result matches the structure of oxy-1a included in Luciola cruciate luciferase.
The structures of oxy-1a–e were optimized while maintaining the conformation of 1a–e
13

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(Figure 4).
Figure 4. The structures of 1a–e with the lowest heat of formation and the
corresponding oxyluciferin form oxy-1a–e.
S →S transitions of all compounds consist of HOMO-LUMO transitions with
0
1
π-π* character. Energy gaps (ꢁEH−L) between HOMO and LUMO and the transition
wavelengths (λ ) estimated from the transition energies for 1a–e show similar variation
tr
to that of the λ values of 1a–e in methanol (Table 1). That is, the λ values of 1b and 1d
fl
tr
are slightly longer than that of 1a, while the λ values of 1a and 1c are similar to each
tr
other. Morpholino analogue 1e has a blue shifted λ value. The variation in the λ values
tr
tr
of oxy-1a–e has similar tendency to that of 1a–e, while the λ values of oxy-1a–e are
tr
longer than those of 1a–e. Oscillator strengths (f) of the S →S transitions of all
0
1
compounds are around 1.4, predicting that fluorescence emissions from the excited
singlet states of oxy-1a–e efficiently proceed with competing internal conversions by
14

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1
9
following Fermi’s golden rule. Thus, oxy-1a–e are predicted to be good light emitters.
Table 2 Calculation data of 1a–e and oxy-1a–e with DFT and TD-DFT using
B3LYP/6-31+G(d).
a
b
c
d
e, f
Compound
HOMO
eV
LUMO
/eV
ꢁEH−L
/eV
Transitions
λ / nm (f )
Configuration
tr
/
1
1
1
1
1
a
b
c
−5.25
−5.15
−5.37
−5.18
−5.50
−5.54
−5.46
−5.64
−5.47
−5.78
−2.01
3.24
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
S0 → S1
395 (1.40)
401 (1.48)
394 (1.43)
402 (1.48)
487 (1.44)
439 (1.38)
445 (1.46)
440 (1.38)
447 (1.46)
432 (1.39)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
H → L (0.70)
−1.96
−2.10
−1.98
−2.18
−2.65
−2.61
−2.73
−2.63
−2.80
3.19
3.27
3.20
3.32
2.89
2.85
2.91
2.84
2.98
d
e
oxy-1a
oxy-1b
oxy-1c
oxy-1d
oxy-1e
S0 → S1
a
b
*
Energy difference between HOMO and LUMO. The π,π transition to the excited singlet state with
c
d
e
the lowest excitation energy. Wavelengths estimated from transition energies. Oscillator strength.
f
Configuration of excitation. H and L denote the HOMO and LUMO, respectively.
In the BL spectra of 1b, 1d and 1e, the minor emission bands were observed at
around 560 nm. A possible explanation of the blue shifted emission is that the excited
oxyluciferin form has a structure with a broken π-electronic conjugation in the
luciferase active site. We looked for a single bond in the structure of oxy-1a, whose
twist effectively affects the π-electronic property, and found that the rotation of the
single bond between the dimethylamino group and the phenyl ring leads to the greatest
15

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changes in the energy levels of HOMO and LUMO (Table S4). The ꢁEH-L value largely
increased with an increase of the twist angle of the C H −NMe bond, due to the
6
4
2
decrease of electron donating effect of the dimethylamino group. Therefore, we predict
that the blue shifted minor emissions of 1b, 1d and 1e originate from the twists of the
cyclic amino groups in the luciferase active site.
2
.4. BLI Properties of AkaLumine 1a, its Analogues 1b–e and LH2.
The whole-body BLI experiments were carried out with transgenic mice
expressing Ppy luciferase by intraperitoneal (IP) injection of 1a–e along with LH . The
2
image pictures were obtained by a BL in vivo imaging instrument using three
independent mice (n=3). In vivo BLI pictures are shown in Figures 5A, S4 and S5.
Overall, total luminescence intensity of the whole-body BLI in mice with 1b was
approximately 2-fold stronger than that with 1a, while the intensities with 1c–e were
weak (Figure 5B). Time courses of BLI intensities with 1b–e show rise and decay
behaviors similar to that with 1a (Figure 5C). The strong intensity with 1b corresponds
to the finding that the L-L reaction with 1b showed higher Φ value than that with 1a.
bl
In the BLI pictures obtained with 1b and 1c, other parts of the bodies from the IP
administered abdomens also showed luminescence, while the BLI picture with LH2
16

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showed localized luminescence around the IP administered abdomen. This result
indicates that 1b and 1c have higher vascular permeability more than LH , leading to
2
whole body distribution of 1b and 1c (Figure S6). In general, permeability and
2
0
distribution of a medicine correlate with lipo-hydrophilic character log P. This
correlation is applicable to the AkaLumine analogues used in this research (Table S16).
Interestingly, however, 1d showed the weakest BLI intensity (4%), while the
L-L reaction with 1d showed a similar Φ value to that with 1a. As a result of cell based
bl
assays with 1a–e and LH using HeLa cells expressing Venus-Ppy (Figure S7 and Table
2
S17), L-L reaction of 1d gave a BL emission spectrum with the maximum only at 567
nm, which is different from the BL spectrum shown in Figure 1. Therefore, the L-L
reactions with 1d under in vivo conditions mainly show blue shifted emissions which
are effectively absorbed by tissues, resulting in the low BLI intensity.
17

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Figure 5. (A) Whole-body BLI of maximum intensity with analogues 1a–e and LH (1
2
mM, 100 ꢀL) into Venus-Ppy transgenic mice. (B) Relative total BL intensity and (C)
time course of BL intensity of whole-body in mice. Data are shown as mean ± SEM (n
=
3).
3
. Conclusion
We newly synthesized AkaLumine analogues 1b
–
e with the cyclic amino
groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl and morpholino groups) and
investigated their BL and BLI properties. The λ values of the major emission bands of
bl
all analogues are in the red region around 670 nm similar to that of 1a, and analogues
1
b, 1d and 1c showed blue-shifted minor emission bands at around 560 nm. The Φbl
18

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values and the kinetic data (K , rel. V
and k ) showed characteristic variations
cat
m
max
depending on the structure of the substituents. In particular, 1-pyrrolidinyl analogue 1b
showed the highest Φ value and 1-azepanyl analogue 1d showed the largest rel. kcat
bl
value among 1a and its analogues. The molecular sizes, hydrophobicities and
flexibilities of the cyclic amino groups in 1b-e modulate the interactions with the
luciferase active site, leading to finely tuned BL properties including emission
wavelength, enzymatic reaction rate and BL quantum yield. The λ values of 1b
–
e were
evaluated by comparison to their fluorescence properties and by correlating to the
π-electronic properties of the corresponding oxyluciferin forms oxy-1b e obtained by
DFT and TD-DFT calculations. The results of BLI with 1a e indicate that 1-pyrrolidinyl
bl
–
–
analogue 1b with the high Φ value gave the brightest imaging picture, indicating that
bl
an improvement of BL properties directly contributes to improvement of BLI properties.
Along with a fine-tuning of the molecular structures of luciferin analogues, it would be
needed to optimize mutant luciferases for a more desirable BL property.
4
. Experimental section
.1. General
4
Commercially available reagents and solvents were used without further
19

ACCEPTED MANUSCRIPT
purification. AkaLumine (1a) was provided by Kurogane Kasei Co., Ltd. For in vitro
bioluminescence measurements, and recombinant Ppy luciferase (QuantiLum
recombinant luciferase, Promega, USA) and Ppy luciferase purified from natural
fireflies (Sigma) were used. A GTA buffer consisting of 0.05 M 3,3-dimethylglutaric
acid, 0.05
M
2-amino-2-hydroxymethyl-1,3-protanediol and 0.05
M
and
2
-amino-2-methyl-1,3-propanediol was prepared and adjusted to pH 8.0 with a NaOH
aqueous solution. For analytical thin layer chromatography (TLC), Wako Silica gel 70
F254 TLC plates were used. Kanto Chemical Silica gel 60 N (spherical, neutral) and
Waters Sep-Pak C18 (ODS, 6 cc Vac Cartridge, 1 g sorbent per cartridge, 55–105 µm
particle size) were used for column chromatography. pcDNA3 vector, which was
purchased from Invitrogen, was used for mammalian expression.
Melting points were measured with a Yamato MP-21 apparatus. IR spectra
1
13
were obtained with a Nicolet 6700 spectrometer with an ATR attachment. H and C
1
NMR spectra were recorded on a JEOL ECA-500 instrument (500 MHz for H and 125
1
3
MHz for C). High-resolution electro-spray ionization (ESI) mass spectra were
obtained with a JEOL JMS-T100LC mass spectrometer. Optical purities of AkaLumine
analogues were analyzed by a Thermo Fisher Scientific LCQ Fleet LC-MS instrument
using a chiral column (Daicel Chemical Industries, OZ-RH, 5 ꢀm, 4.6–150 mm) with a
20

ACCEPTED MANUSCRIPT
linear gradient of 10% to 90% acetonitrile in H O over 30 min (flow rate 0.3 mL/min)
2
as an eluent, and were detected with a UV detector at 330 nm. UV/visible absorption
spectra were obtained with a Varian Cary 60 spectrophotometer (scan speed, 600
nm/min; data interval, 1 nm). Fluorescence spectra and fluorescence quantum yields
were measured with a Hamamatsu Photonics Quantaurus-QY absolute PL quantum
yields measurement system. Bioluminescence intensities were monitored using an
ATTO AB-2200 luminometer, and bioluminescence spectra were measured with an
ATTO AB-1850 spectrophotometer (data interval, 1 nm). LogP and ClogP calculations
were performed with ChemDraw 13.0. Density functional theory (DFT) calculations
2
1
were performed with the Gaussian 09 program (Rev. D.01). DFT includes the B3LYP
2
2
function with the 6-31G(d) basis set. Molecular graphics were made with GaussView,
2
3
Version 5. The BLI was measured with a multifunctional in vivo imaging system
(MIIS, Molecular device, LLC.).
4
4
1
3
.2. Synthesis of AkaLumine analogues 1b–e
.2.1. 4-(azepan-1-yl)benzaldehyde (2d). A solution of 4-fluorobenzaldehyde (105 μL,
.0 mmol), hexamethyleneimine (224 μL, 2.0 mmol) and potassium carbonate (415 mg,
.0 mmol) in DMF (0.5 mL) was stirred at 80 °C under Ar for 16 h. The product was
21

ACCEPTED MANUSCRIPT
extracted with a mixture of ethyl acetate (EtOAc) and hexane (1:4) (2 x 50 mL). The
organic layer was washed with brine, dried over Na SO , and concentrated in vacuo.
2
4
The residue was purified by flash silica gel column chromatography (hexane/EtOAc
1
[
4:1]) to give 2d (134 mg, 0.66 mmol, 66%) as orange oil; H NMR (500 MHz, CDCl )
3
δ 9.69 (s, 1 H), 7.70 (d, J = 9.2 Hz, 2 H), 6.70 (d, J = 9.2 Hz, 2 H), 3.52 (t, J = 6.0 Hz,
1
3
4
1
2
H), 1.79 (br. s, 4 H), 1.55–1.54 (m, 4 H); C NMR (125 MHz, CDCl ) δ 190.0, 153.4,
3
+
32.3, 124.7, 110.6, 49.6, 27.2, 26.7; HR-ESI-MS: m/z: [M+H] calcd for C H NO,
1
3
18
04.1388; found, 204.1408.
4
.2.2. Ethyl (2E,4E)-5-(4-(pyrrolidin-1-yl)phenyl)penta-2,4-dienoate (3b). To a solution
of 2b (1.75 g, 10 mmol) and triethyl 4-phosphonocrotonate (3.3 mL, 15 mmol) in THF
40 mL), sodium hydride (60% dispersion in mineral oil, 800 mg, 20 mmol) was
(
carefully added at 0 °C under Ar. After the reaction mixture was stirred at ambient
temperature for 3 h, the reaction was quenched by adding ethanol (1 mL) and water (20
mL), and the product was extracted with EtOAc (3 x 20 mL). The organic layer was
washed with brine, dried over Na SO , and concentrated in vacuo. The residue was
2
4
purified by silica gel column chromatography (hexane/EtOAc [4:1]) to give 3b (1.9 g,
-1
7
.0 mmol, 70%) as yellow crystals: mp 138–141 °C; IR (neat, ν, cm ): 1698, 1588,
22

ACCEPTED MANUSCRIPT
1
1
234, 1001; H NMR (500 MHz, CDCl )
δ
7.44 (dd, J = 11.2, 15.4 Hz, 1 H), 7.34 (d, J
8.6 Hz, 2 H), 6.82 (d, J = 15.5 Hz, 1 H), 6.66 (dd, J = 11.2, 15.2 Hz, 1 H), 6.51 (d, J =
.2 Hz, 2 H), 5.85 (d, J = 15.5 Hz, 1 H), 4.21 (q, J = 7.3 Hz, 2 H), 3.31 (m, 4 H), 2.00
3
=
9
13
(
m, 4 H), 1.30 (t, J = 7.2 Hz, 3 H); C NMR (125 MHz, CDCl ) δ 167.6, 148.4, 146.0,
3
1
41.5, 128.8, 123.3, 121.0, 117.6, 111.6, 60.0, 47.5, 25.4, 14.4; HR-ESI-MS: m/z: [M +
+
Na] calcd for C H NO Na, 294.1470; found, 294.1483.
1
7
21
2
Diene ester 3c–e were prepared with a similar procedure to the preparation of
3
b.
Ethyl (2E,4E)-5-(4-(piperidin-1-yl)phenyl)penta-2,4-dienoate (3c) (yield 85%),
-1
1
yellow crystals: mp 108–109 °C; IR (neat, ν, cm ): 1698, 1593, 1117, 1000; H NMR
(
500 MHz, CDCl ) δ 7.43 (dd, J = 11.4, 15.4 Hz, 1 H), 7.33 (d, J = 8.6 Hz, 2 H), 6.85 (d,
3
J = 8.6 Hz, 2 H), 6.81 (d, J = 15.5 1 H), 6.70 (dd, J = 10.8, 15.4Hz, 1 H), 5.89 (d, J =
1
1
1
5.5 Hz, 1 H), 4.21 (q, J = 6.9 Hz, 2 H), 3.23 (m, 4 H), 1.68 (m, 4 H), 1.60 (m, 2 H),
1
3
.30 (t, J = 6.8 Hz, 3 H); C NMR (125 MHz, CDCl ) δ 167.5, 152.4, 145.6, 140.9,
3
28.6, 126.2, 122.8, 118.9, 115.4, 60.2, 49.6, 25.6, 24.4, 14.5; HR-ESI-MS: m/z: [M +
+
H] calcd for C H NO , 286.1807, found 286.1806.
1
8
24
2
Ethyl (2E,4E)-5-(4-(azepan-1-yl)phenyl)penta-2,4-dienoate (3d) (yield 72%),
-1
yellow crystals: mp 83–85 °C; IR (neat, ν, cm ): 1699, 1585, 1518, 1233, 1117, 998;
23

ACCEPTED MANUSCRIPT
1
H NMR (500 MHz, CDCl ) δ 7.44 (dd, J = 15.2, 11.2 Hz, 1 H), 7.34 (d, J = 8.6 Hz, 2
3
H), 6.82 (d, J = 15.5 Hz, 1 H), 6.67 (m, 3 H), 5.86 (d, J = 14.9 Hz, 1 H), 4.21 (q, J = 7.1
Hz, 2 H), 3.49 (t, J = 6.0 Hz, 4 H), 1.80 (s, 4 H), 1.54 (s, 4 H), 1.31 (t, J = 7.2 Hz, 3 H);
1
3
C NMR (125 MHz, CDCl ) δ 167.7, 149.7, 146.1, 141.5, 129.1, 123.3, 121.2, 117.8,
3
1
11.3, 60.1, 49.4, 27.6, 27.0, 14.5; HR-ESI-MS: m/z: [M + H]+ calcd for C H NO ,
19 26 2
3
00.1964; found, 300.1976.
Ethyl (2E,4E)-5-(4-morpholinophenyl)penta-2,4-dienoate (3e) (yield 76%),
-1
1
yellow crystals: mp 128–130 °C; IR (neat, ν, cm ): 1700, 1596, 1243, 1116, 1008; H
NMR (500 MHz, CDCl ) δ 7.42 (dd, J = 10.9, 15.5 Hz, 1 H), 7.36 (d, J = 8.6 Hz, 2 H),
3
6
.84 (d, J = 8.6 Hz, 2 H), 6.81 (d, J = 16.0 Hz, 1 H), 6.71 (dd, J = 10.9, 15.5Hz, 1 H),
.90 (d, J = 14.9 Hz, 1 H), 4.20 (q, J = 7.1 Hz, 2 H), 3.83 (m, 4 H), 3.19 (m, 4 H), 1.30
5
1
3
(
t, J = 6.8 Hz, 3 H); C NMR (125 MHz, CDCl ) δ 167.4, 151.7, 145.4, 140.5, 128.6,
3
+
1
27.5, 123.5, 119.5, 115.1, 66.8, 60.3, 48.5, 14.5; HR-ESI-MS: m/z: [M + H] calcd for
C H NO , 288.1600; found, 288.1589.
17
22
3
4
.2.3.
Methyl
N-((2E,4E)-5-(4-(pyrrolidin-1-yl)phenyl)penta-2,4-dienoyl)-S-trityl-
D
-cysteinate (4b).
To a solution of 3b (1.35 g, 5 mmol) in 2-propanol (20 mL), water (20 mL) and 5 M
24

ACCEPTED MANUSCRIPT
NaOH aq. (5 mL) were added under Ar, and the mixture was heated under reflux for 3 h.
The reaction mixture was neutralized by adding 5 M HCl aq. (5 mL). The precipitates
were collected by suction filtration, washed with water and dried in vacuo to give
(
2E,4E)-5-(4-(pyrrolidin-1-yl)phenyl)penta-2,4-dienoic acid. The dienoic acid was dried
under reduced pressure and used in the next reaction. To a solution of the diene acid in
DMF (20 mL), -Cys(S-Trt)-OMe (1.9 g, mmol) and
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (1.45 g, 7.5
D
5
1
mmol) were added at ambient temperature under Ar, and the reaction mixture was
stirred at 30 °C for 24 h. After adding water (20 mL) to the mixture, the product was
extracted with EtOAc (200 mL x 2). The combined organic layer was washed with brine,
dried over Na SO , and concentrated in vacuo. The residue was purified by silica gel
2
4
column chromatography (hexane/EtOAc [2:1]), to give 4b (1.6 g, 2.6 mmol, 52%) as
-1
1
yellow crystals: mp 94–102; IR (neat, ν, cm ): 1738, 1645, 1583, 1483, 1139; H NMR
500 MHz, CDCl3) 7.39–7.18 (comp., 18 H), 6.80 (d, J = 14.9 Hz, 1 H), 6.65 (dd, J =
0.9 14.9 Hz, 1 H), 6.50 (d, J = 8.6 Hz, 2 H), 6.02 (d, J = 8.0 Hz, 1 H), 5.81 (d, J = 14.9
(
δ
1
13
Hz, 1 H), 4.74 (m, 1 H), 3.70 (s, 3 H), 3.30 (m, 4 H), 2.70 (m, 2 H), 2.00 (m, 4 H); C
NMR (125 MHz, CDCl3) 172.3, 166.1, 148.4, 144.4, 143.2, 141.0, 129.6, 128.8,
δ
25

ACCEPTED MANUSCRIPT
1
28.1, 127.0, 123.7, 121.2, 119.8, 111.8, 67.0, 52.7, 51.2, 47.7, 34.2, 25.6; ESI-MS:
+
m/z: [M + Na] 625.20.
Diene amides 4c–e were prepared with a similar procedure to the preparation
of 4b.
(S)-1-methoxy-1-oxo-3-(tritylthio)propan-2-yl
(2E,4E)-5-(4-(piperidin-1-yl)phenyl)penta-2,4-dienoate (4c) (yield 60%), yellow
-1
1
crystals: mp 86–89; IR (neat, ν, cm ): 1738, 1650, 1589, 1510, 1487; H NMR (500
MHz, CDCl3) 7.39–7.19 (comp., 18 H), 6.86 (d, J = 8.6 Hz, 2 H), 6.79 (d, J = 15.5 Hz,
H), 6.69 (dd, J = 10.9 15.5 Hz, 1 H), 6.05 (d, J = 8.0 Hz, 1 H), 5.84 (d, J = 14.9 Hz, 1
H), 4.74 (m, 1 H), 3.70 (s, 3 H), 3.23 (m, 4 H), 2.70 (m, 2 H), 1.68 (m, 4 H),1.60 (m, 4
δ
1
1
3
H); C NMR (125 MHz, CDCl )
δ
171.2, 165.9, 152.3, 144.4, 142.8, 140.2, 129.6,
3
1
28.4, 128.1, 127.0, 126.5, 122.8, 120.9, 115.5, 67.0, 52.8, 51.2, 49.7, 34.2, 25.6, 24.4;
+
+
ESI-MS: m/z: [M + H] 617.27, [M + Na] 639.25.
Methyl
N-((2E,4E)-5-(4-(azepan-1-yl)phenyl)penta-2,4-dienoyl)-S-trityl-D-cysteinate
(4d)
-1
(
yield 45%), yellow crystals: mp 86–89; IR (neat, ν, cm ): 1738, 1651, 1584, 1515,
1
1
487, 1182, 1141; H NMR (500 MHz, CDCl ) δ 7.44–7.35 (m, 6 H), 7.36–7.30 (m, 3
3
H), 7.30–7.24 (m, 6 H), 7.21 (t, J = 7.2 Hz, 3 H), 6.79 (d, J = 15.5 Hz, 1 H), 6.69–6.59
26

ACCEPTED MANUSCRIPT
(
(
(
m, 3 H), 5.98 (d, J = 7.4 Hz, 1 H), 5.81 (d, J = 14.9 Hz, 1 H), 4.83–4.65 (m, 1 H), 3.71
1
3
s, 3 H), 3.48 (t, J = 6.0 Hz, 4 H), 2.70 (m, 2 H), 1.79 (m, 4 H), 1.55 (m, 4 H); C NMR
125 MHz, CDCl ) δ 171.3, 166.1, 149.6, 144.5, 143.3, 140.8, 129.7, 129.0, 128.2,
3
1
27.0, 123.6, 121.2, 119.8, 111.3, 67.0, 52.8, 51.2, 49.4, 34.3, 27.7, 27.1; ESI-MS: m/z:
+
[
M + Na] 653.22.
(S)-1-methoxy-1-oxo-3-(tritylthio)propan-2-yl
(2E,4E)-5-(4-morpholinophenyl)penta-2,4-dienoate (4e) (yield 62%), yellow crystals:
-1
1
mp 85–87; IR (neat, ν, cm ): 1732, 1650, 1591, 1511, 1236, 1191; H NMR (500 MHz,
CDCl ) δ 7.39–7.19 (comp., 18 H), 6.86 (d, J = 8.6 Hz, 2 H), 6.80 (d, J = 15.5 Hz, 1 H),
3
6
.72 (dd, J = 10.9 15.5 Hz, 1 H), 6.09 (d, J = 8.0 Hz, 1 H), 5.87 (d, J = 14.9 Hz, 1 H),
13
4
.74 (m, 1 H), 3.85 (m, 4 H), 3.71 (s, 3 H), 3.20 (m, 4 H), 2.70 (m, 2 H); C NMR (125
MHz, CDCl ) δ 171.2, 165.8, 151.6, 144.4, 142.6, 139.8, 129.6, 128.4, 128.1, 127.7,
3
+
1
6
27.0, 123.5, 121.4, 115.2, 67.0, 66.8, 52.8, 51.2, 48.6, 34.2; ESI-MS: m/z: [M + Na]
41.24.
4
.2.4.
Methyl
(S)-2-((1E,3E)-4-(4-(pyrrolidin-1-yl)phenyl)buta-1,3-dien-1-yl)-4,5-dihydrothiazole-4-c
arboxylate (5b). To a solution of trifluoromethanesulfonic anhydride (Tf O) (347 μL,
2
27

ACCEPTED MANUSCRIPT
2
.12 mmol) in CH Cl (10 mL), 4b (640 mg, 1.06 mmol) in dry CH Cl (5 mL) was
2
2
2
2
added at 0 °C under Ar, and the reaction mixture was stirred for 30 min. After adding
saturated NaHCO aq. (20 mL) to the reaction mixture, the product was extracted with
3
CHCl (3 x 20 mL). The combined organic layer was washed with brine, dried over
3
Na SO , and concentrated in vacuo. The residue was purified by silica gel column
2
4
chromatography (hexane/EtOAc [2:1]), to give 5b (72.6 mg, 0.21 mmol, 21%) as
-1
1
orange crystals: mp 147–149; IR (neat, ν, cm ): 1713, 1592, 1519, 1153, 984; H NMR
500 MHz, CDCl3) 7.33 (d, J = 8.6, 2 H), 6.93 (dd, J = 10.9, 14.9 Hz, 1 H), 6.75 (d, J
15.5 Hz, 1 H), 6.68 (dd, J = 10.9, 15.5 Hz, 1 H), 6.51 (d, J = 14.9 Hz, 1 H), 6.49 (d, J
8.6 Hz, 2 H), 5.15 (t, J = 9.2 Hz, 1 H), 3.82 (s, 3 H), 3.59–3.49 (comp., 2 H), 3.30 (m,
(
δ
=
=
4
1
3
H), 1.99 (m, 4 H); C NMR (125 MHz, CDCl )
δ
171.6, 170.3, 148.4, 144.1, 140.0,
3
1
28.8, 123.6, 122.2, 121.9, 111.8, 77.9, 52.9, 47.6, 34.6, 25.6; HR-ESI-MS: m/z: [M +
+
H] calcd for C H N O S, 343.1480; found, 343.1509.
1
9
23
2
2
Thiazoline esters 5c–e were prepared with a similar procedure to the
preparation of 5b.
Methyl
(S)-2-((1E,3E)-4-(4-(piperidin-1-yl)phenyl)buta-1,3-dien-1-yl)-4,5-dihydrothiazole-4-ca
-1
rboxylate (5c) (yield 42%), orange crystals: mp 118–119 °C; IR (neat, ν, cm ): 1737,
28