Molecular mechanism of Symplectoteuthis bioluminescence - Part 4: Chromophore exchange and oxidation of the cysteine residue

Article abstract of DOI:10.1016/j.bmc.2014.05.044

Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-coelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH's of the cysteine residues via conjugate addition reaction. This

Full text of DOI:10.1016/j.bmc.2014.05.044

Accepted Manuscript
Molecular Mechanism of Symplectoteuthis Bioluminescence---Part 4: Chromo-
phore Exchange and Oxidation of the Cysteine Residue
Chun-Ming Chou, Yu-Wen Tung, Minoru Isobe
PII:
S0968-0896(14)00402-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.044
BMC 11605
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2014
20 May 2014
21 May 2014
Please cite this article as: Chou, C-M., Tung, Y-W., Isobe, M., Molecular Mechanism of Symplectoteuthis
Bioluminescence---Part 4: Chromophore Exchange and Oxidation of the Cysteine Residue, Bioorganic & Medicinal
Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.044
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Molecular Mechanism of Symplectoteuthis
Bioluminescence---Part 4: Chromophore
Exchange and Oxidation of the Cysteine Residue
Leave this area blank for abstract info.
Chun-Ming Chou, Yu-Wen Tung, Minoru Isobe
Chemistry Department, National Tsing Hua University, 101, Sec. 2, Kuang Fu Road, Hsinchu, 30013, Taiwan.
Binding site
Cys
Binding site
Cys
Binding site
Cys
Binding site
Cys
S
S
Active site
390Cys
Active site
390Cys
S
S
S
S
S
S
Cys
Cys
Cys
Cys
Binding site
Binding site
S
S
Binding site
Increase of fluorescence
intensity at 520 nm.
Binding site
Decrease of fluorescence
intensity at 520 nm.
11 (4-OH-DCL-bn)
15 (2,4-FF-DCL-nap)
-
-
+H3N
+H3N
CO2
CO2
R2
O
S
R2
S
R2
O
O
20 eq. L-Cysteine
pH=6.0
O2
N
N
N
R4
bn=
N
N
N
N
NH
R8
pH=7.8
R4
R4
R8
R8
N
H
HO
HO
HO
nap=
11 (4-OH-DCL-bn), R2=H, R4=OH.
15 (2,4-FF-DCL-nap), R2=R4=F.
35 (Cys-4-OH-DCL-bn), R2=H, R4=OH. fluorescence at 520 nm.
19 (Cys-2,4-FF-DCL-nap), R2=R4=F. NO FLUORESCENCE at 520 nm.
0

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Molecular Mechanism of Symplectoteuthis Bioluminescence---Part 4: Chromophore
Exchange and Oxidation of the Cysteine Residue
Chun-Ming Chou, Yu-Wen Tung, Minoru Isobe
Chemistry Department, National Tsing Hua University, 101, Sec. 2, Kuang Fu Road, Hsinchu, 30013, Taiwan. minoru@mx.nthu.edu.tw
ARTICLE INFO
ABSTRACT
Article history:
Received
Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-
coelenterazine (DCL) at the binding sites and the active site. This binding takes place through
the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the
chromophore molecules at the binding cites first, and then moves to the active cite Cys-390 for
the luminescence. The current study focuses on these dynamic aspects of the chromophore
using the natural photoprotein by analyzing the fluorescence changing of the DCL
chromophores analogs with 8-(4’-methoxyphenyl)- or 8-(2’-naphthyl)-group and 2-(2’,4’-
difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at
slightly acidic media and also from the luminescence function observed at the optimum pH 7.8.
The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at
pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid
chromatographic detection from the spent solutions.
Received in revised form
Accepted
Available online
Keywords:
Bioluminescence
Symplectin
coelenterazine
photoprotein
2009 Elsevier Ltd. All rights reserved.
1. Introduction
dehydro-coelenterazine analog; thus, a peak corresponding to
MW 843.55 as the evidence of the coelenterazine analog being
This article is the continuation of Part 3 of our previous report
on Symplectoteuthis bioluminescence.1
bound to the active site before the luminescence. Another peak
equivalent to MW 831.53 was observed after the luminescence
from the tryptic digest as the coelenteramide analog.5 This
detection system includes a house-modified capillary-HPLC
connected to ESI-Q-TOF/MS on the samples from the
photoprotein, which was reconstructed with synthetic
chromophore and then hydrolyzed with proteolytic enzyme such
as Trypsin or Lys-C.5 Further proof of the active site was
implemented using di-fluorinated coelenterazine, which binds to
the same 390-Cys residue more tightly. It gave the molecular
ions of the chromopeptide after luminescence at m/z 849.3 as
well as the fragment ions (m/z 735, 588, 419, 306 and 278) from
the capillary HPLC-ESI-Ion Trap MS to show the coelenteramide
substructure.1
Light emission from living cells has been commonly used in
chemical biology research as one of the important tools for
specific visualization of the target molecules on the basis of
molecular-binding in living cells. Coelenterazine is the most
typical chromophore in marine bioluminescent systems, and
recently 2-hydroperoxycoelenterazine is the key feature in the
calcium dependent photoproteins such as aequorin and obelin.
These studies have made the molecular mechanism of the
bioluminescence by means of structural biology using X-ray
analysis of the co-crystallized photoproteins with the substrates.2
We have been working on the coelenterazine-containing
photoprotein symplectin, the luminescence of which is triggered
by monovalent ions such as Na+ and K+.3 This photoprotein
symplectin works in an oceanic squid, Symplectoteuthis
oualaniensis (Japanese name, TOBI-IKA) collected in the sea
around Okinawa, Japan,4 and obtainable in a water-insoluble
fraction but only soluble with high salt-concentration, e.g. KCl
>0.6 mol/L.4 In 2008, the amino acid sequence of symplectin
was elucidated with its 501 AA containing 11 cysteine residues
(including 3 Cys-S-S-Cys). The current study was carried out
using the purified photoprotein from the squid since gene-
manipulated preparation of this protein has not been reported.
Isobe et al assigned that the 390-Cys worked as the active site of
symplectin by means of LC-MS-detection and the following
experiments.5 Namely, the chromo-peptide fragment, chrom-S-
390C-G-L-393K, was observed by using the mono-fluorinated
Shimomura states that many marine bioluminescent animals
give light by using coelenterazine or similar compounds as the
substrate having common chromophore.6
The oxidation
mechanism and peroxy intermediates of these chromophores
show the common substructures as the hydroperoxide 5 and
peroxylactone (dioxetanone) 6,6 as has been demonstrated by
Isobe and co-workers to prove the more detailed mechanism in a
direct manner using 100%-13C-enriched luminous coelenterazine
analogs at some specific carbon atoms and analyses through low-
temperature 13C-NMR of the short-life time peroxidic
intermediates (Figure 1).7
The presence of 2-
hydroperoxycoelenterazine in aequorin and obelin has been
reported recently.2, 8
1

Cys390
Cys390
Cys390
O2
-O
S
S
S
O
O
N
N
O
O
O
*
*
*
*
N
*
N
O2, K+, pH=7.8
O
N
N
N
N
OH
Coelenterazine
OH
OH
K+
K+
N
H
peroxide
intermediate
dioxetanone
intermediate
HO
HO
HO
1
2
3
δ126.8
HO
O
O
O
5
3
2
O
δ169.5
δ139.2
O
CO2
O
δ152.5
δ107.9
warm-up
photo oxygenation
O
N
N
δ178.7
N
N
NH
Bn
N
NH
N
N
δ182.6
δ157.9
CF3CD2OD-CD3OD
-78°C
δ108.7
δ139.5
δ108.1
δ109.8
R6
N
H
Bn
R6
R6
N
Bn
R6
N
Bn
R6=
HO
5
7
4
6
Figure 1. The possible bioluminescence intermediates 1 and 2 of symplectin (top) were proved through synthesizing a t-butyl model 4, having 100%-13C
enriched coelenterazine and the corresponding hydroperoxide 5 and peroxy lactone 6 and assigning the structures by low-temp NMR (bottom).7
PPh3
HO
O
O
N
N
NH2
Bn
O
MeOH
O
N
N
N
N
NH
R6
O=PPh
+
R6
Bn
3
R6
N
Bn
5a
8
9
Ph
MeOH
PPh3
Ph
O
Ph
O
N
N
O
P
O
N
N
NH2
Bn
O
NH
Bn
O
N
NH
Bn
R6
R6
+ O=PPh3
R6
N
6a
10
9
Figure 2. Titrative reduction of the peroxide 5a and dioxetanone 6a were effected by triphenyl phosphine at -78 °C to diminish the light emission (Usami
and Isobe.6).
In this case, those reactive intermediates were prepared via
low-temperature photo-oxygenation in CF3CD2OD solvent. The
NMR at -78 °C proved the structures of 2-hydroperoxy-
coelenterazine analog 5 and dioxetanone 6 with concomitant
measurements of luminescent spectra from these intermediates
under different media. Namely, 5 and 6 provided significant
luminescence spectra derived not only from neutral media but
also even from acidic media by simply allowing the temperature
at ca -50 °C or 0 °C, respectively. This provided new tools
elucidating the excited-molecular species, which is strong
contrast to the fact that previous chemi-luminescence had been
reported only under strongly basic conditions in dipolar aprotic
solvents such as DMSO. Titrative reduction of the peroxides 5
and 6 at -78 °C with triphenyl phosphine or diphenyl sulfide took
place in the stoichiometry-dependent manner with diminishing
the amount of luminescence; (triphenyl phosphine was as
example as shown in Figure 2).7 The products in the reduction
photoprotein aequorin.5, 9 Involvement of the sulfur atom of Cys
residues provided new tools for the studies of bioluminescence,
which is described in the current study.
Kongjinda and Isobe1 reported that two di-fluoro-
dehydrocoelenterazine regio-isomers (FF-DCL-bn: deep red
color, non-fluorescent) 12 and 13 showed quite different
bioluminescence behavior in symplectin photoprotein from that
of natural chromophore 11 (4-OH-DCL-bn). Namely, 2,4-
difluoro-analog 12 (2,4-FF-DCL-bn) provides larger amount of
the luminescent light than the natural chromophore 11, while 2,6-
difluoro-analog 13 (2,6-FF-DCL-bn) gives smaller amount
(1/3~1/4) of light from 11.9 Both of these two chromophores 12
and 13, however, showed similar behavior such as increase of the
green fluorescence at 520 nm during the incubation to apo-
symplectin at pH 6.0 in accordance with the binding as a
conjugate addition manner of the SH group of Cys to these
chromophores (Figure 5). When the pH of the DCL-analog-
incubated photoproteins was changed to pH 7.8, both analogs
show decreasing of the green fluorescent intensities. Namely, the
non-bioluminescent species from 13 was consumed as well like
the luminescent one 12. This suggested some different pathways
might take place for each of them on the protein surface when
subjected to the luminescence conditions.
were amino-pyrazines
9 and triphenylphosphine oxide or
diphenyl sulfoxide. So the bioluminescence mechanism should
include the same chemical principle on the enzymic surface.
Recently, Isobe et al. further reported the fact that two different
bioluminescence systems including sulfur atom of DTT
(dithiothreitol) attached at the cysteine residues of the
2

F
F
2'
3
2
O
O
O
4'
6'
N
N
N
N
N
N
N
N
5
OH
F
F
6
8
N
HO
HO
HO
F
F
F
O
O
O
O
11 (4-OH-DCL-bn)
12 (2,4-FF-DCL-bn)
13 (2,6-FF-DCL-bn)
N
N
N
N
N
N
N
N
N
N
N
OH
F
F
F
N
OMe
HO
HO
HO
HO
14 (4-OH-DCL-nap)
15 (2,4-FF-DCL-nap)
16 (2,6-FF-DCL-nap)
17 (2,4-FF-DCL-pmp)
Figure 3. Natural (11, 4-OH-DCL-bn) and unnatural (12-17) chromophores applied to apo-symplectin photoprotein.
The synthetic route of the coelenterazine-chromophores10 was
redesigned to avoid using hazardous diazo-reagents as reported
by Kishi et al.11 and the improved version reported by Kondo et
al.12 Alternative routes on the basis of Pd-mediated cross
coupling13 was also developed by Makarasen and Isobe,14 and
Wong and Isobe.15 Chou, one of the authors of this paper, has
published a new synthetic route from aminopyrazine, which was
suitable for the synthesis of the current study.16 In this paper, we
prepared such chromophores 11-17 (see Figure 3) via the new
synthetic route that have different fluorescence maxima and
intensities emerging after binding with the cysteine residues.
These compounds led us to have the first experimental
achievement of the chromophore-exchange on the photoprotein,
symplectin, from originally bound-natural chromophore 11 to the
new difluoro-derivatives 12, 13, 15, and 16. Of particular
interests, 15 (2,4-FF-DCL-nap) and 16 (2,6-FF-DCL-nap)
derivatives did not exhibit strong fluorescence at 520 nm. We
have confirmed these observations with symplectin by using
various sample preparations which yield different amounts of
apo-symplectin.
solution for providing active oxygen species. The light amounts
were integrated for 5 min, and the resulting values were
compared as the light yields, which were almost identical (
3
%) from the same sample-preparation obtained from the same
individual squid organ (data not shown). Incubation of each
three different chromophore-DCL-analog 11, 12, and 13 in
DMSO solution (2 mM, 4 ꢀL) with the symplectin-solution (40
ꢀL) was implemented at 0 °C for 20 min, followed by
luminescence measurement by mixing the luminescence buffer
and catalase solution at 25 °C. Each chromophore was incubated
with three different symplectin-solutions P-1, P-2 or P-3 prepared
from the organs of different squids. (In fact, 10 symplectin-
solutions were prepared from the organs of 10 different squids,
and typical three examples (photoprotein P-1, P-2 and P-3) are
illustrated in Figure 4.)
2. Results and Discussions
2.1. Preparation of the symplectin solution
A partially purified (SDS-PAGE single band) sample solution
of symplectin was prepared on the basis of its limited solubility
upon KCl-concentration according to our previously reported
method with slight modification.1 Every symplectin-preparation
was prepared from the frozen-photogenic organ by cutting as a
round disk by using a 1.5 cm-diameter borer to give a disk-
weight of 50 mg ( 2 mg). The disk was homogenized with a
glass-homogenizer using the basic buffer (containing 0.4M KCl,
pH=7.8 , 1000 ꢀL) and washed by washing buffer A (containing
0.4M KCl with DTT, pH=5.6, 1000 ꢀL) once and washing buffer
B (containing 0.4M KCl without DTT, pH=5.6, 1000 ꢀL) twice.
Figure 4. Typical results of the light amounts from three different
symplectin/apo-symplectin photoprotein (P-1, P-2 and P-3) after incubating
with different chromophores; thus, DMSO (containing no chromophore), and
containing 4-OH-DCL (11), 2,4-FF-DCL (12), or 2,6-FF-DCL (13) solution;
first in buffer pH 5.6 for 20 min and then the light amounts were counted by
changing the pH to 7.8 and averaged from three measurements.
In Figure 4, the control experiments by adding only DMSO
solvent afforded different amounts of light (76%, 66% and 46%)
with the photoprotein P-1, P-2, and P-3, respectively (see Figure
4/DMSO). These results suggest that the photoprotein contains
luminous chromophore in different amounts in the photoprotein,
which is considered to remain as storage form but in different
quantities. In fact, the incubation with the natural chromophore
11 (4-OH-DCL-bn) afforded the results of the same light-
production in these three photoprotein preparations P-1, -2, -3, so
these values are plotted as 100% as the full capacity of
symplectin with the natural chromophore. Namely, the ratios of
apo-symplectin:holo-symplectin can be determined on each
individual squid as [(100-DMSO)/DMSO], thus, concluding that
photoprotein P-1 has 24%, and P-2 has 35% and P-3 has 54% of
the apo-protein, respectively.
The residue was extracted with the extracting buffer
A
(containing 0.6M KCl, pH=5.6, 1000 ꢀL). Each photoprotein
preparation was kept in extracting buffer A at pH 6.0 which
contained sucrose without DTT at 0 °C in an ice bath placed in a
refrigerator ca. 4 °C. All of them were used within 18 hrs for the
following experiments.
2.2. Bioluminescence activity
Each given sample was placed in a short test tube in a dark
small house equipped within a photon counter and the light
amount was measured by repeating three times and averaged.
First, bioluminescence light amount from each aliquot (40 ꢀL) of
symplectin solution was measured (without incubation with any
additional synthetic chromophore) under a pH 7.8 environment
by simply adding the luminescence buffer (400 ꢀL) and catalase
3

A.
-
+H3N
CO2
S
bn=
nap=
pmp=
F
F
F
F
O
O
O
O
L-Cysteine
N
N
NH
R8
N
N
N
N
N
N
N
tBuOK
F
F
F
F
DMSO
R8
N
H
R8
N
H
R8
HO
HO
HO
HO
12, 2,4-FF-DCL-bn
15, 2,4-FF-DCL-nap
17, 2,4-FF-DCL-pmp
18, Cys-2,4-FF-DCL-bn
19, Cys-2,4-FF-DCL-nap
20, Cys-2,4-FF-DCL-pmp
24, 2,4-FF-amide-bn
25, 2,4-FF-amide-nap
26, 2,4-FF-amide-pmp
21, 2,4-FF-CL-bn
22, 2,4-FF-CL-nap
23, 2,4-FF-CL-pmp
OMe
B.
Figure 5. (A) Synthetic analogs showing different property in UV absorbance (12, 15, and 17) and fluorescence (18, 19, and 20).
(B) Fluorescence spectra of the three chromophores 18 (10 ꢀM), 19 (100 ꢀM), and 20 (100 ꢀM), and three coelenteramides, which are the luminescence
product as known as oxyluciferin, 24 (20 ꢀM), 25 (20 ꢀM), and 26 (20 ꢀM).
The yellow bars and blue bars in Figure 4 represent the light
amounts observed after incubation with 12 (2,4-difluoro-
dehydrocoelenterazine; 2,4-FF-DCL-bn), and with 13 (2,6-
difluoro-dehydrocoelenterazine; 2,6-FF-DCL-bn). The results of
the light yields from the chromophore 12 were increased by
+23%, +50% and +68% with P-1, P-2 and P-3, respectively.
Meanwhile, the corresponding amounts from 13 decreased by -
33%, -49% and -72%. These results (the increase or decrease
figures) proposed a new question whether it might be simply
related to the amount of apo-symplectin due to those figures
being somewhat similar; thus, 24%, 35% and 54%. What is the
reason by increase or decrease of the luminescence activity under
the same operation with different chromophores 12 and 13.
Therefore, we have designed some experiments to observe in
more details (1) whether simple intake of the chromophore takes
place at the vacant binding sites or (2) whether any chromophore
exchange takes place from natural DCL with unnatural di-FF-
DCL. To urge such experiments onward, we synthesized three
(p-OMe)-phenyl derivatives in the following experiments,
because the resulted bioluminescence product 25 (2,4-FF-amine-
nap) has the fluorescence peak at 441 nm away from that of 24
(2,4-FF-amide-bn) showing at 415 nm. Informatively, the
fluorescence of 26 (2,4-FF-amide-pmp) has the similar peat at
423 nm as that of 24. A conjugate addition of a cysteine-SH
residue to DCL can readily demonstrate in vitro as shown in
Figure 6, where red color of DCL changes to pale yellow color
of CL by stepwise addition of cysteine into the chromophores.
Such equilibria with these difluoro DCL derivatives were
measured to provide the dissociation constants as KD50= 2.8x10-4
M for 12, as KD50= 1.3x10-3 M for 15, and KD50= 5.3x10-4 M for
17, with clear isosbestic points at 410 nm and 500 nm showing
direct equilibria between these chromophores and Cysteines as in
Figure 6. A decreasing KD50 value of the naphthyl analog 19 may
be attributed to a repulsive interaction of larger and non-flexible
naphthyl group than the benzyl group of the cysteine adducts 18.
And KD50 values of 18 and 20 were similar. Cysteine adducts 19
and 20 should, however, showed enough binding affinity to
investigate the reaction with apo-symplectin.
naphthyl chromophores 14, 15 and 16 as well as
a
methoxyphenyl chromophore 17 displacing the benzyl group at
the 8-position of the imidazopyrazinone nucleus. In fact, the 8-ꢁ-
naphtyl and 8-(p-OMe)-phenyl derivatives did not show
fluorescence at 520 nm as the 8-benzyl derivatives.
Similar equilibria may be observed through changing the
fluorescent intensity at 520 nm when the synthetic
chromophores, eg. 12 (2,4-FF-DCL-bn) and 15 (2,4-FF-DCL-
nap), were incubated to the (apo)-symplectin preparations. In
case of the 8-benzyl DCL derivatives 12, such an exchange
highly exceeds the natural coelenterazine 11 due to the presence
of 2 electron-withdrawing F-atoms.
2.3. Dynamic Profile of the Chromophore Exchange by using
2,4-diF-DCL-naphtyl
The different fluorescent nature between the green fluorescent
coelenterazine and non-fluorescent dehydrocoelenterazine is
striking, although UV-Vis spectra behaves similar but different
wave length as CL is pale yellow color but DCL shows deep red
color (Figure 5). The weak fluorescence of 19 (Cys-2,4-FF-
DCL-nap, 602 nm) and 20 (Cys-2,4-FF-DCL-pmp, 600 nm) are
much different than 18 (Cys-2,4-FF-DCL-bn, 522 nm). However,
8-ꢁ-naphtyl derivatives were selected as a better analog than 8-
In order to confirm the fact that incorporation of naphthyl-
DCL analog 15 indeed takes place into apo-symplectin, the other
DCL and its analogs (shown in Figure 7) were incubated, one by
one, to apo-symplectin at pH 5.6 and raised to pH 7.8 to compare
the bioluminescence activities. The relative light yields are
illustrated in Figure 7.
4

Figure 6. UV absorbance spectrum of the equilibrium DCL and analog with Cysteine. The DCL samples were dissolved in degassed DMSO, and then
mixed with pH 6 buffer (10 mM KCl, 12 mM AcOH, 12 mM NaOAc in 68% MeOH/H2O. The volume ratio of DMSO to buffer is 1 : 2.) in the UV cell by
shaking. a) x eq. L-Cysteine, pH 6 buffer.
Figure 7. The bioluminescence activity of DCL and analogs. DMSO and chromophore were incubated with the apo-symplectin which was extracted from the
same squid for 20 mins, and then measured luminescent light yield.
5

Figure 8. The fluorescence change of symplectin with DCL and analogs incubation (A). The plotting at 520 nm of fluorescence change (B).
Figure 9. The fluorescence change at 520 nm of the double incubation with 2 different DCL analogs. The 12 (2,4-FF-DCL-bn) and 15 (2,4-FF-DCL-nap)
have similar binding affinity in symplectin because the fluorescence intensity at 520 nm remained in similar level while the competitive experiment.
The synthetic naphthyl chromophores 15 (2,4-FF-DCL-nap)
and 16 (2,6-FF-DCL-nap) showed higher light yield than 11 (4-
OH-DCL-bn), but 14 (4-OH-DCL-nap) didn’t. 17 (2,4-FF-DCL-
pmp) also had similar function as 15 (2,4-FF-DCL-nap).
According to the results, the synthetic analog 15 (2,4-FF-DCL-
nap) has similar binding affinity and bioluminescence as 11 (4-
OH-DCL-bn).
decreasing-intensity when natural 4-OH-DCL-bn on symplectin
was displaced by 15 (2,4-FF-DCL-nap).
After 30 min, the reconstructed symplectin was treated with
basic buffer (pH=11) to change to pH 7.8. So these base treated
solutions should consume the chromophore (pseudo-reduced
coelenterazines) to form coelenteramides with concomitant light
emission so both solutions showed the decrease at 520 nm
(Figure 8B). It is worth to mention that the fluorescence didn’t
become 0 even the chromophores were consumed by adding of
basic buffer. The possible reason was due to the coelenteramide
which has 10% of fluorescence intensity at 520 nm compared to
(pseudo-reduced chromophore) coelenterazine.
The time-dependent fluorescence spectrum-changes are shown
in Figure 8. This measurement was implemented by sucking the
reconstructed symplectin-solution into a flow cell equipped with
a fluorometer at the 5-10 min intervals from 0 min to 30 min
after starting the incubation. The increase of the FL intensity at
520 nm with 12 (2,4-FF-DCL-bn) was due to the binding to a
cysteine residue of symplectin to form an apparently-reduced
coelenterazine chromophore with the green fluorescence.
However, the decrease of the FL intensity at 520 nm after the
incubation with 15 (2,4-FF-DCL-nap) was due to the fact that the
cysteine adduct (the same chromophore as 19) has only 1%
intensity (compared to 18)(see Figure 5B) to result in the
A competitive experiment is shown in Figure 9. It is
observed that 15 (2,4-FF-DCL-nap) binds stronger with cysteine
residue of symplectin than 11 the natural 4-OH-DCL-bn. This is
concluded from the observation of decreasing 520 nm intensity
by incubation with 15 (2,4-FF-DCL-nap). This is consistent
from the KD50 values between the reconstructed symplectin
preparations with 11 (4-OH-DCL-bn) (see Figure 7).
6

Binding site
Cys
Binding site
Cys
A.
apo-symplectin
Binding site
Cys
Binding site
Cys
S
S
Active site
390Cys
Active site
390Cys
HS
S
S
S
S
S
Cys
Cys
Cys
Cys
Binding site
Binding site
HS
S
Binding site
Binding site
Increase of fluorescence
intensity at 520 nm.
Binding site
Cys
Binding site
Cys
S
11 (4-OH-DCL-bn)
12 (2,4-FF-DCL-bn)
15 (2,4-FF-DCL-nap)
Active site
390Cys
S
S
S
Cys
Cys
Binding site
Binding site
S
Decrease of fluorescence
intensity at 520 nm.
Binding site
Cys
Binding site
Cys
B.
apo-symplectin
Binding site
Cys
Binding site
Cys
S
S
Active site
390Cys
Active site
390Cys
HS
S
S
S
S
S
Cys
Cys
Cys
Cys
Binding site
Binding site
Binding site
HS
S
Binding site
Increase of fluorescence
intensity at 520 nm.
Binding site
Cys
Binding site
Cys
S
Active site
390Cys
S
S
S
Cys
Cys
Binding site
Binding site
S
Same fluorescence
intensity at 520 nm.
Figure 10. The possible exchange happened in symplectin with double incubation by 2 different DCL analogs.
(A) The chromophores in 11 (4-OH-DCL-bn) incubated symplectin were easy exchanged by 15 (2,4-FF-DCL-nap) and the fluorescence intensity at 520 nm is
◆
decreasing as blue line(-- --) as shown in Figure 9.
(B) The pink line (--ꢂ--) in Figure 9 shows the 12 (2,4-FF-DCL) incubation and followed by 15 (2,4-FF-DCL-nap).
Figure 11. Color and fluorescence change of symplectin with different incubation. The 11 (4-OH-DCL-bn) or 12 (2,4-FF-DCL-bn) chromophore incubated
symplectin showed the fluorescence under UV irradiation, but 15 (2,4-FF-DCL-nap) or 17 (2,4-FF-DCL-pmp) didn’t.
7

Figure 12. Fluorescence intensity-changes by different chromophores incubated with two apo-symplectin A and B (retaining more natural chromophore).
And the corresponding bioluminescence light amounts in pH 7.8.
Figure 13. Color difference in chemiluminescence and bioluminescence of 12 (2,4-FF-DCL-bn) and 15 (2,4-FF-DCL-nap).
The relative binding affinity maybe similar strength between
12 (2,4-FF-DCL-bn) and 15 (2,4-FF-DCL-nap) in symplectin
judging from the intensities at 520 nm remained similarly after
the 2nd incubation. Based on these results, 15 (2,4-FF-DCL-nap)
is recognized by symplectin with stronger binding affinity than
natural chromophore, and also shows bioluminescence. Such a
difference in the fluorescence intensities at 520 nm provides
good evidence for the chromophore-exchange to take place in
symplectin (see Figure 10).
To confirm the result, the naked eye observations are shown in
Figure 11 by monitoring the fluorescence of the reconstructed
symplectin under UV light (365 nm) with various synthetic
chromophores for 20 min-incubation.
There is obvious
difference in the colors between before and after the incubation
with 12 (2,4-FF-DCL-bn) or 15 (2,4-FF-DCL-nap). Only the
incubation of 11 (4-OH-DCL-bn) and 12 (2,4-FF-DCL-bn) to
symplectin shows the fluorescence at 520 nm, but the ones from
15 (2,4-FF-DCL-nap) and 17 (2,4-FF-DCL-pmp) do not.
8

Figure 14. Fluorescence spectra and chromatograms and of bioluminescence product, which were extracted from the symplectin after the incubation with 12
(2,4-FF-DCL-bn) (A) or 15 (2,4-FF-DCL-nap) (B).
The remaining 11 (4-OH-DCL-bn) in symplectin solution
could affect the light yield of bioluminescence. The larger rest
amount of 4-OH-DCL-bn led the change of fluorescence at 520
nm more obviously (Figure 12B). To prove the luminescence is
indeed resulted from 15 (2,4-FF-DCL-nap), the bioluminescence
was recorded by digital camera. For the incubation of 12 (2,4-FF-
DCL-bn), it is obviously that the mono-anion intermediate is the
light emitter in the bioluminescence compared to
chemiluminescence (Figure 13). For the incubation of 15 (2,4-
FF-DCL-nap), the bioluminescence was different to the mono-
anion or dianion intermediate in chemiluminescence. It is
possible that 15 (2,4-FF-DCL-nap) would form neutral
intermediate to result in the bioluminescence of symplectin. The
anion structures of natural coelenterazine and coelenteramide are
discussed by Shimomura and Teranishi.8
(amine-nap) at 17 min, respectively, from the 15 (2,4-FF-DCL-
nap) incubated symplectin.
3. Conclusions
The fluorescence change by the incubation to symplectin
provided us a good evidence to prove the chromophore, which
bound to the binding sites of symplectin, can exchange with the
outcome synthetic chromophore depended on the relative binding
affinity to cysteine residue. Because 19 (Cys-2,4-FF-DCL-nap)
only shows weak fluorescence at 600 nm, this characteristic
nature makes the observation much easier to confirm the
decreasing of the fluorescence at 520 nm, which is represented
by coelenterazine. Those results indicate the effect of fluorinated
modification on the coelenterazine lead to different
bioluminescence activity. And also the ratios of apo-symplectin :
holo-symplectin can be determined, such as 35% of apo-
symplectin in the protein solution and 65% of holo-symplectin in
Figure 8, and 91% of apo-symplectin in the protein solution and
9% of holo-symplectin in Figure 12A.
N
N
NH2
N
N
NH2
HO
HO
27, amine-bn
28, amine-nap
The result is also provided us that the light yield of 12 (2,4-
FF-DCL-bn) and 13 (2,6-FF-DCL-bn) in symplectin is due to the
directly reaction between the chromophore and symplectin. And
the decrease of light yield from 13-(2,6-FF-DCL-bn)-incubated
symplectin may be caused by the intermolecular oxidation on
Cysteine390 while the pseudo-reduced coelenterazine 2 was
formed.
Figure 15. Structure of 35 (amine-bn) and 36 (amine-nap)
Because of the fact that no direct evidence of 15 (2,4-FF-
DCL-nap) results in light emission with symplectin, we try to
search for the luminescence product instead. The spent solution
of nap-symplectin solution was extracted with dichloromethane,
and then analyzed by HPLC (Figure 14). Comparison of the
chromatograms (retention time and the fluorescence intensity) of
the related peaks of luminescence product coelenteramide 24 and
25, and coelenteramine 35 and 36 (the structure was shown in
Figure 15), it is obvious to find coelenteramide 24 (2,4-FF-
amide-bn) was found in the extraction from the incubation of 12
(2,4-FF-DCL-bn) to symplectin, while the correlated peaks were
found coelenteramide 25 (2,4-FF-amide-nap) at 70 min and 36
9

dried over MgSO4 and then concentrated under reduced
pressureto obtain diacyl compound. The diacyl compound was
dissolved in MeOH/dioxane (15 mL/22 mL) and 1N NaOH(aq)
(5.6 mL) was added. After stirring at rt for 1.5 h, the reaction was
stopped by adding 1N HCl(aq) (5.6 mL). Water (50 mL) was
added and the solution was extracted with EtOAc (3 x 20 mL).
The combined organic layer was washed with brine, dried over
MgSO4 and then concentrated under reduced pressure. The
residue was purified by silica column chromatography with
EtOAc in hexane. The product is a mixture of coelenteramide
and bis-acylated compound. It was then recrystallized by
dissolving in hot acetone and adding 50% EtOAc in hexane to
afford amide 2-21 only as a white crystal.
4. Experimental
Instruments: Fluorescence spectra were measured with a
JASCO FP-2020+ spectrometer and Hitachi F-4500
spectrometer. Bioluminescence was obtained on a Hamamastu
Photonic Multi-channel Analyzer, PMA-11 by simultaneous
integration of light with different wavelengths (300-600 nm)
without scanning against the wavelength. Total amounts of the
luminescent light were acquired with Gene Light GL-200S to
obtain the relative light yield. Centrifugation was performed by
using a Hettich Mikro 120 centrifuge machine. Fluorescence
spectra were recorded and analyzed by Spectra Manager version
2.07.02. 1H NMR spectra were measured at Varian MR-400 (400
1
MHz) and Varian Mercury-400 (400 MHz). H NMR chemical
4.1.1.1. N-(3-benzyl-5-(4-hydroxyphenyl)pyrazin-2-
yl)-2-(2,4-difluorophenyl)acetamide (24)
shifts are referenced to the CHCl3 singlet (7.24 ppm) and the
center of DMSO quintet (3.30 ppm). Data are reported as follows;
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q
= quartet, qn = quintet, sep =septet, br = broadened, m =
multiplet), coupling constant(s), assignment, and integration,
respectively. 13C NMR spectra were measured at Bruker NMR
DMX-600 (125 MHz), Varian MR-400 (100 MHz) and Varian
Mercury-400 (100 MHz). 13C NMR chemical shifts are
referenced to the center of the CDCl3 triplet (77.0 ppm) and the
center of DMSO septet (39.5 ppm). High resolution mass spectra
(HRMS) were obtained by Finnigan/ Thermo Quest MAT 95XL
mass spectrometer in NCHU, Varian 901-MS/ TQ- FT mass
spectrometer and MAT-95XL HRMS in NTHU. High resolution
mass spectra (HRMS) are reported in m/z.
1
Yield: 50%; H NMR (500 MHz, DMSO-d6): ꢃ 3.76 (2H, s),
4.11 (2H, s), 7.04-7.08 (3H, m), 7.16-7.27 (6H, m), 7.43 (1H, q,
J= 8.3 Hz), 8.03 (2H, d, J= 8.7 Hz), 8.88 (1H, s), 10.54 (1H, s)
ppm. 13C NMR (125 MHz, DMSO-d6): ꢃ 103.6 (t, J= 26 Hz),
111.2 (dd, J= 21, 3 Hz), 114.4, 118.9 (dd, J= 16, 3 Hz), 126.3,
128.0, 128.3, 129.0, 133.1 (dd, J= 9, 6 Hz), 137.2, 138.3, 143.5,
148.3, 150.5, 160.6, 160.7 (dd, J= 246, 12 Hz), 161.4 (dd, J=246,
12 Hz), 168.9 ppm. 19F NMR (470 MHz, DMSO-d6): -112.8,
-112.5 ppm. HRMS (APCI): calcd for C26H22F2N3O2 (M+H+)
432.1445; found 432.1674.
4.1.1.2. 2-(2,4-difluorophenyl)-N-(5-(4-
hydroxyphenyl)-3-(naphthalen-2-yl)pyrazin-2-
yl)acetamide (25)
Chemicals:
dihydrogenphosphate
Potassium chloride (KCl), Potassium
(KH2PO4), Dipotassuium
Yield: 46%. 1H NMR (500 MHz, DMSO-d6): ꢃ3.85 (2H,
s),4.16 (2H, s), 7.43 (1H, q, J= 8.5 Hz), 7.56-7.58 (2H, m), 7.91-
8.01 (5H, m), 8.07 (2H, d, J= 8.4 Hz), 8.98 (1H, s), 10.64 (1H, s)
ppm. 13C NMR (125 MHz, DMSO-d6): ꢃ 103.7 (t, J= 26 Hz),
111.2 (dd, J= 21, 3 Hz), 114.4, 118.9 (dd, J= 16, 3 Hz), 125.1,
125.7, 126.2, 126.3, 128.3, 128.5, 128.7, 133.1 (dd, J= 9, 6 Hz),
132.3, 134.6, 138.3, 143.5, 148.3, 150.5, 160.6 (dd, J= 245, 13
Hz), 161.7 (dd, J= 245, 13 Hz), 167.9 ppm. ppm. 19F NMR (470
MHz, DMSO-d6): -112.6, -112.8 ppm. HRMS (APCI): calcd for
C28H19F2N3O2 (M+H+) 468.1447; found 468.1578.
hydrogenphosphate (K2HPO4), Ethylenediaminetetraacetic acid
(EDTA), and 30% Hydrogen Peroxide (H2O2) were purchased
from SHOWA. 99% L-Cysteine and sucrose were purchased
from ACROS. Dithiothreitol was purchased from Baker
Analyzed. Dimethyl sulfoxide (DMSO) was purchased from
Baker. Catalase (Catalase from bovine liver, 2000-5000 units/mg
protein) was purchased from Sigma-Aldrich.
UV absorbance measurement of Cys-DCL adducts: DMSO
and pH 6 buffer (10 mM KCl, 12 mM AcOH, 12 mM NaOAc in
68% MeOH/H2O) were degassed by Ar purging and then stored
under Ar atmosphere. The sample was dissolved in degassed
DMSO, and then mixed with pH 6 buffer (the volume ratio of
DMSO to buffer is 1 : 2.) in the UV cell or fluorescence cell. For
UV measurements, the cysteine solution was added in dropwise
to the cell, mixed by shaking. The UV spectra were measured
with different amount of L-cysteine.
4.2. Preparation of apo-symplectin solution
Luminous organ (50 mg)
basic buffer, 1 mL x 1.
homogenize at 25 °C, incubate 15 mins
at 25 °C, and then incubation on ice for 1h.
centrifuge 105g, 10 mins, 4 °C.
basic buffer: 50mM K2HPO4, 250mM sucrose,
0.4M KCl, 1mM DTT, 1mM EDTA, pH=7.8.
O2 bubble before using.
4.1. Synthesis of coelenterazine, coelenteramide, coelenteramine
and analogs
ppt
washing buffer 1, 1 mL x 1.
centrifuge 105g, 10 mins, 4 °C.
washing buffer A: 50mM KH2PO4, 250mM sucrose,
1mM DTT, 1mM EDTA, 0.4M KCl, pH=5.6.
washing buffer B: 50mM KH2PO4, 250mM sucrose,
0.4M KCl, pH=5.6.
Coupound 11-17, 24, 27, and 28 were reported in previous
.
study1,16,17
ppt
extracting buffer A: 50mM KH2PO4, 250mM sucrose,
0.6M KCl, pH=5.6
extracting buffer B: 50mM KH2PO4, 250mM sucrose,
1.0M KCl, pH=5.6
4.1.1. General method to synthesize coelenteramide
washing buffer 2, 1 mL x 2.
centrifuge 105g, 10 mins, 4 °C.
2,4-difluorophenylacetic acid (320 mg, 1.86 mmol) dissolve in
thionyl chloride (1.3 mL) was heated to reflux for 2 h. Vapors
ppt
(SO2 and HCl) generated were trapped with saturated NaHCO3(aq)
The solution was cooled to room temperature, evaporated under
reduced pressure (distillation in large scale), and dried under
vacuo. Crude 2,4-difluorophenylacetyl chloride 2-15 was
obtained as a colorless oil. To a solution of coelenteramine
(0.186 mmol) in anhydrous CH2Cl2 (0.4 mL) and pyridine (1 mL)
was added solution of 2,4-difluorophenylacetyl chloride in
CH2Cl2 (0.6 mL) slowly at 0 ˚C and stirred for 5 h. Then the
solution was poured into sat. Na2HCO3(aq) (80 mL), extracted
with CH2Cl2. The combined organic layer was washed with brine,
.
12h, 4 °C.
extracting buffer A or B, 1 mL x 1.
centrifuge 105g, 10 mins, 4 °C.
supernatant
(apo-symplectin A or B)
ppt
The yellow luminous organ of a squid Symplectoteuthis
oualaniensis (kept frozen at -78 °C) was cut in each 50 mg using
a borer into a round disk of 15 mm diameter.
10

4.4.1. Fluorescence spectrum of single incubation
at pH=6. 0.
For each experimental preparation one disk was homogenized
with 1 mL basic buffer (50 mM potassium phosphate, 250 mM
sucrose, 1 mM DTT, 1 mM EDTA, pH 8.0, the buffer being
saturated with O2 by bubbling before use) at 25 °C for 15 min.
This operation allowed consumption of the remaining natural
chromophore in the homogenate. Each homogenate was then
cooled to 4 °C for 1 h and centrifuged at 10,100 G at 4 °C for 10
min to yield the photoprotein in precipitation. This precipitate
was washed once with washing buffer A (50 mM KH2PO4, 250
mM sucrose, 1 mM DTT, 1 mM EDTA, 0.4 M KCl, pH= 5.6),
and twice with washing buffer B (50 mM KH2PO4, 250 mM
sucrose, 0.4 M KCl, pH= 5.6). The photoprotein was extracted
from the washed precipitates with extracting buffer A (50 mM
KH2PO4, 250mM sucrose, 0.6 M KCl, pH= 5.6) or extracting
buffer B (50 mM KH2PO4, 250 mM sucrose, 1.0 M KCl, pH=5.6)
to obtain an apo-Symplectin solution A with buffer A, or solution
B with buffer B, respectively. The apo-Symplectin A and B was
kept at 4 °C for 12 h, and used for the following experiments.
In these chromophore exchange experiments, apo-symplectin
solutions were prepared from the same squid in the incubation
step. And each sample was sucked into the flow cell to record
the spectrum as 0 min. Each photoprotein solution was drained
back to an eppendorf tubing and incubated with a chromophore
either of 2,4-FF-DCL-bn, 2,4-FF-DCL-nap, or necessary DCL as
a control such as 4-OH-DCL-bn. The repeated measurements
were applied after 5, 10, and 20, 30 minutes to compare the
fluorescence change with different incubation time.
4.4.2. Fluorescence spectrum of double incubation
at pH=6. 0.
In the incubation and comparison step the relative binding
affinity of synthetic DCL to symplectin, apo-symplectin solutions
were prepared from the same squid, and operated as the same as
above. Thus obtained reconstructed-symplectin solutions were
further incubated with DCL or analogs for comparison and
sucked into the flow cell to record the spectra and plotted at 520
nm against these operations to analyze the relative binding
affinity.
4.3. Measurements of fluorescence spectra:
apo-symplectin solution (400 µL)
4.4.3. Fluorescence spectra of consumption of the
chromophores.
measure fluorescence spectrum
To obtain the fluorescence change with the chromophores
consumption, the reconstructed symplectin (as prepared in B)
was treated with 100 ꢀl basic buffer (50mM K2HPO4, 0.6M KCl,
1mM EDTA, pH=11.) to adjust the pH value to 7.8. This
operation leads symplectin to give bioluminescence (reported by
Vorawan et al; and photograph Fig. 7 and 10.). The fluorescence
spectra were recorded at 5, 10, 20 min, and recorded as 35, 40, 50
min.
16µL 2mM DCL or analogs in DMSO
kept at 4 °C, 5 mins.
measure fluorescence spectrum (0 min).
reconstructed symplectin
kept at 4 °C, 5 mins.
measure fluorescence spectrum
repeat 2 times (5 min) (10 min).
kept at 4 °C, 10 mins.
measure fluorescence spectrum.
repeat 2 times (20 min) (30 min).
4.4.4. Comparison
add 100µL pH=11 buffer, kept at rt, 5 mins.
measure fluorescence spectrum.
repeat 2 times (35 min) (40 min).
To compare the fluorescence change, the intensity at 520 nm
of each spectrum was plotted into the diagram intensity at 520
nm via time.
kept at rt, 10 mins.
measure fluorescence spectrum (50 min).
4.5. Measurements of integrated light amount from
apo-symplectin A (100 µL)
Each reconstructed symplectin preparation was prepared in a
similar way by addition of 16 ꢀL 2 mM DCL in DMSO into 400
ꢀL apo-symplectin solution as above. The fluorescence spectra
were measured with a JASCO FP-2020 spectro-fluorescence
detector equiped with a flow cell (size, 20 ꢀL capacity) by
sucking the 50 ꢀL sample solution into the dry cell with a
microcyrinder. Each fluorescence spectrum was recorded from 0
min (before incubation), 5 min, 10 min, 15 min, 20 min, and 30
min. Then a 100 ꢀL pH=11 buffer (50 mM K2HPO4, 0.6 M KCl,
1 mM EDTA, pH= 11) was add to the reconstructed symplectin
solution to change the pH to 7.8 in 30 min, and the fluorescence
spectra were recorded at 35 min, 40 min, and 50 min.
4 µL 2mM DCL or analogs in DMSO
kept at 4 °C, 20 mins.
catalase: 1 mg catalase dissolved
in 1 mL extracting buffer A.
reconstructed symplectin
lumi buffer: 50mM K2HPO4, 0.6M KCl,
1mM EDTA, pH=8.
500 µL H2O2 was added to
15 mL buffer before using.
reconstructed symplectin (40 µL)
1. add 20 µL catalase.
2. add 400 µL luminescence buffer.
3. measure bioluminescence.
bioluminescence
The reconstructed symplectin was prepared by addition of 4
ꢀL synthetic chromophore solution (2 mM DCL in DMSO) to
100 ꢀL apo-Symplectin solution A at 4 °C, and kept for 20 min
for incubation. The bioluminescence, each total integrated light
was measured by simultaneous mixing of the following three
solutions; thus, (1) 20 ꢀL catalase solution (1 mg catalase
dissolved in 1 mL extracting buffer A), (2) 400 ꢀL luminescence
buffer (50 mM K2HPO4, 0.6 M KCl, 1 mM EDTA, pH= 8), and
(3) 500 µL H2O2 was added to 15 mL buffer before using). This
mixture was added to a 40 ꢀL reconstructed symplectin solution,
which was placed in a dark cell equipped with a photo counter.
The light amounts were integrated for 5 min from each sample
preparation with a Gene Light GL-200S photo counter. Relative
integrated-light amounts were compared each other.
4.4. Fluorescence measurement of reconstructed symplectin
The reconstructed symplectin was prepared by the addition of
16 ꢀL 2 mM DCL in DMSO to 400 ꢀL apo-symplectin
preparation. The reconstructed symplectin solution was sucked
into the flow cell of JASCO FP-2020 spectrometer. The
fluorescence spectra were recorded and analyzed by a Spectra
Manager software-version 2.07.02.
Before the each
measurement, the extracting buffer A (50 mM KH2PO4, 250mM
sucrose, 0.6 M KCl, pH=6.0) was sucked into the flow cell to
wash the remained photoprotein in the cell.
11

4.6. Chemiluminescence measurement
References and notes
To a 0.6 mL 2 mM solution of coelenterazine analogs in
anhydrous DMSO was added 1 N t-BuOK in t-BuOH. The
chemiluminescence was recorded by a digital camera or a
Hamamastu Photonic Multi-channel Analyzer, PMA-11 by
simultaneous integration of light with different wavelengths
(300-600 nm) without scanning against the wavelength.
1.
For the Part 3, see V. Kongjinda, Y. Nakashima, N. Tani, M. Kuse, T.
Nishikawa, C.-H. Yu, N. Harada, and M. Isobe, Chem. Asian J. 2011, 6,
2080-2091. In this paper, the “chromophore(s)” mean(s) the precursor,
dehydrocoelenterazine (imidazopyrazinone), and the “substrate(s)”
mean(s) the “chromophore” both before and after the covalently bound
form (dihydroimidazopyrazinone) with the cysteine residue of the
photoprotein, simplectin.
2.
(a) E. V. Eremeeva, L. P. Burakova, V. V. Krasitskaya, A. N.
Kudryavtsev, O. Shimomura, and L. A. Frank, Photochem. Photobiol.
Sci. 2014, 13, 541-547; (b) E. V. Eremeeva, S. V. Markova, W. J. H. v.
Berkel, and E. S. Vysotski, J. Photochem. Photobiol. B: Biology 2013,
127, 133-139.
F. I. Tsuji, and G. B. Leisman, Proc. Natl. Acad. Sci. USA 1981, 78,
6719-6723.
H. Takahashi, and M. Isobe, Bioorg. Med. Chem. Lett. 1993, 3, 2647-
2652.
4.7. Analysis of bioluminescence product
The bioluminescence product was extracted by CH2Cl2 from
the symplectin solution which was finished bioluminescence,
concentrated by vacuum, and then dissolved in MeOH for HPLC
analysis. The HPLC was purchased from Jasco. The pump is
Jasco PU-2080. The UV detector is UV-2075+, and the
fluorescence detector is FP-2020+. The sample was analyzed by
45% MeCN containing 0.1% TFA in ODS-HG-5 column (4.6 x
250 mm) with 0.5 mL/min flow rate. The UV detector was set for
350 nm absorbance. The fluorescence detector is recorded 520
nm (em) which is with 350 nm excited irradiation.
3.
4.
5.
6.
M. Isobe, M. Kuse, N. Tani, T. Fujii, and T. Matsuda, Proc. Jpn. Acad.,
Ser. B 2008, 84, 386-392.
(a) Shimomura, O. Bioluminescence, Chemical Principles and
Methods, 2006, World Scientific Publishing C. Pte. Ltd., Hackensack,
NJ 07601, USA. (b) Isobe, M.; Kuse, M.; Fujii, T.; Usami, K.
"Landmarks in Phytobiology: Proceedings of the 12th International
Congress on Photobiology" ICP '96, Ed. by Hoeningsmann, H. et al.,
1998, 149-156.
4.8. Fluorescence measurement of synthetic compound
7.
(a) M. Isobe, H. Takahashi, K. Usami, M. Hattori, and Y. Nishigohri,
Pure Appl. Chem. 1994, 66, 765-772; (b) K. Usami, and M. Isobe,
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Tetrahedron 1996, 52, 12061-12090; (d) K. Usami, and M. Isobe,
Chem. Lett. 1996, 25, 215-216.
DMSO and pH 6 buffer (10 mM KCl, 12 mM AcOH, 12 mM
NaOAc in 68% MeOH/H2O) were degassed by Ar purging and
then stored under Ar atmosphere. The sample was dissolved in
degassed DMSO, and then mixed with pH 6 buffer (the volume
ratio of DMSO to buffer is 1 : 2.) in a UV cell or a fluorescence
cell. The final concentration was 2 ꢀM for coelenteramine, 10
ꢀM for coelenteramide. For fluorescence measurements, the
cysteine solution was added to cell, mixed by shaking. The
fluorescence spectrum was measured as soon as possible after
mixing to avoid the air oxidation to result coelenteramine.
8.
9.
J. F. Head, S. Inouye, K. Teranishi, and O. Shimomura, Nature 2000,
405, 372-376.
I. Doi, M. Kuse, T. Nishikawa, and M. Isobe, Bioorg. Med. Chem.
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10. S. Inoue, S. Sugiura, H. Kakoi, K. Hasizume, T. Goto, and H. Iio,
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11. O. Shimomura, and B. M. Y. Kishi, Biochem. J. 1989, 261, 913-920.
12. (a) N. Kondo, M. Kuse, T. Mutarapat, N. Thasana, and M. Isobe,
Heterocycles 2005, 65, 843-856; (b) M. Kuse, N. Kondo, Y. Ohyabu,
and M. Isobe, Tetrahedron 2004, 60, 835-840.
13. (a) For introducing 8-position group of DCL or CL: M. Adamczyk, S.
R. Akireddy, D. D. Johnson, P. G. Mattingly, Y. Pan, and R. E. Reddy,
Tetrahedron 2003, 59, 8129-8142; (b) For introducing 6-position and
the 8-position group of DCL or CL: M. Mosrin, T. Bresser, and P.
Knochel, Org. Lett. 2009, 11, 3406-3409.
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Acknowledgments
Financial support from Ministry of Science and Technology
(the previous name is National Science Committee in Taiwan) is
gratefully acknowledged. Authors are also indebted to National
Tsing Hua University for the equipments.
16. C.-M. Chou, Y.-W. Tung, M.-I. Lin, D. Chan, W. Phakhodee, and M.
Isobe, Hetercycles 2012, 86, 1323-1339.
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