Development of simple firefly luciferin analogs emitting blue, green, red, and near-infrared biological window light

Article abstract of DOI:10.1016/j.tet.2013.03.050

Simple firefly luciferin analogs emitting blue, green, and red light were developed. The longest emission maximum was observed at 675 nm, which belongs to the NIR biological window (650-900 nm), useful for deep site bioimaging of living animals. The analogs showed a slow rise of emission intensity compared with the rapid emission of natural luciferin. The light emission of the adenylated analogs was strongly enhanced compared with those of analogs themselves.

Full text of DOI:10.1016/j.tet.2013.03.050

Tetrahedron 69 (2013) 3847e3856
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Development of simple firefly luciferin analogs emitting blue, green,
red, and near-infrared biological window light
Satoshi Iwano, Rika Obata, Chihiro Miura, Masahiro Kiyama, Kazutoshi Hama,
*
Mitsuhiro Nakamura, Yoshiharu Amano, Satoshi Kojima, Takashi Hirano, Shojiro Maki ,
*
Haruki Niwa
Department of Engineering Science, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Simple firefly luciferin analogs emitting blue, green, and red light were developed. The longest emission
maximum was observed at 675 nm, which belongs to the NIR biological window (650e900 nm), useful
for deep site bioimaging of living animals. The analogs showed a slow rise of emission intensity com-
pared with the rapid emission of natural luciferin. The light emission of the adenylated analogs was
strongly enhanced compared with those of analogs themselves.
Received 22 January 2013
Received in revised form 11 March 2013
Accepted 12 March 2013
Available online 18 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Bioluminescence
Firefly
Luciferin analog
Near-infrared
Biological window
1. Introduction
Because of its substrate specificity and high sensitivity, firefly lu-
ciferase and luciferin combination is widely used in the detection of
Firefly luciferase is well known for its highly efficient light
emission by catalyzing the oxidation of substrate -luciferin (
ATP, and biological studies, with luciferase as a reporter gene in cell
culture systems,4 and recently in noninvasive whole-body bio-
imaging.5 For the bioimaging technique, emissions of light with
various colors are useful. In particular, red, or more desirably, the
D
D-
LH2). The accepted catalytic mechanism of firefly bioluminescence
is shown in Fig.1.1e3 This enzymatic light emission process does not
Fig. 1. Proposed reaction catalyzed by firefly luciferase. In the first step of the reaction, luciferase (Luc) catalyzes adenylation of D-LH2 with ATP in the presence of Mg2þ to generate
the intermediate luciferyleAMP (D-LH2eAMP) accompanied by pyrophosphate (ppi) (Eq. 1). Then the oxidative decarboxylation of the intermediate gives excited-state oxyluciferin,
which then releases visible light in the course of relaxation to the ground state (Eq. 2).
require an external light source to fluorescence, thereby an ex-
tremely high signal-to-noise ratio is provided by this process.
light around the 650e900 nm region termed the near-infrared
(NIR) biological window,6 is suitable for noninvasive whole-body
imaging. Since the light of the NIR window is not strongly absor-
bed by oxygenated hemoglobin and melanin in animal tissues, the
light is expected to be useful for deeper site imaging.
* Corresponding authors. Tel.: þ81 42 443 5484; fax: þ81 42 486 1966 (H.N.); tel.:
þ81 42 443 5493; fax: þ81 42 486 1966 (S.M.); e-mail addresses: maki@pc.uec.ac.jp
(S. Maki), niwa@pc.uec.ac.jp (H. Niwa).
In addition to Lampyridae (firefly) luciferases, several different
isozymes are known to emit light in different colors using the same
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.03.050

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S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
substrate D
-LH2.7 Based on a bioengineering study, several mutant
luciferases emitting various colors were developed.8,9 Among var-
ious luciferases including their mutants, the shortest emission
maximum wavelength was 534 nm and the longest was 623 nm.7
Theoretical study of the light emitter oxyluciferin indicated an
expected emission range from natural D
-LH2 to be 421e626 nm.10
This study suggested that a new substrate scaffold, other than the
natural luciferin chromophore, is required in order to obtain
a wider emission range and longer maximum emission wavelength
with Lampyridae luciferases including their mutants.
After the structure of luciferin was elucidated and synthesized in
the 1960s, some modified luciferin analogs, such as 60-amino-
luciferin,11 were synthesized and a few of them functioned as light-
emitting substrates.3 More recently, N-alkylated 60-aminoluciferins
were found to act as substrates12 and cyclic alkylaminoluciferin was
reported to show red light emission (607 nm) by using the mutant
of Photuris pennsylvanica firefly luciferase (Ultra-Glo).13 Some
modified luciferins were newly developed for color change and
specific purposes.14e22 However, most synthetic bioluminescent
luciferin analogs consist of benzothiazole and 4-carboxythiazolin-
2-yl rings unchanged from natural luciferin, except for quinolyl-,
coumaryl-, naphthylluciferins,17,23a and very recently published23b
heterocyclic luciferin analogs.
Fig. 3. Structure around plausible ligand-binding site of P. pyralis luciferase and
overlay model of oxy-3b, oxyluciferin and AMP. The backbone structure of luciferase
(4G36) is shown in cyan, and the electrostatic surface is presented as a wire mesh with
negative and positive charges in red and blue, respectively. The original ligand DLSA is
omitted from the figure for clarity. Side chains of Arg218, Ans229, Phe247, Tyr255,
Ser284, Glu311, and Arg337 are shown in stick form. The view was sliced to show the
binding pocket and some residues were omitted from the figure for clarity. Water
molecules are shown as red spheres, except Wat725 is colored green. The ligand atoms
are displayed in stick form with the carbon atom of oxy-3b in magenta, oxyluciferin in
lemon yellow and AMP in orange. Nitrogen is colored blue, oxygen is colored red,
sulfur is colored yellow, and phosphorus is colored deep orange.
In this study, we have substituted a benzothiazole moiety with
a simplified aromatic structure to investigate the effect of
p con-
jugation to the emission wavelength (Fig. 2).
Fig. 2. Chemical structure of synthetic substrates (1e3).
2. Results and discussion
bottom of the binding pocket and provided the rather negative
electrostatic surface expected in a cation-stabilizing environment.
Notably, the nitrogen atom of oxy-3b closely overlapped the water
molecule (Wat725) of the 4G36 crystal structure. This preliminary
evaluation indicated that it is structurally possible to locate the
designed analogs on the native luciferin binding site.
As a simple bioluminescence chromophore for luciferase sub-
strate, we chose a 4-hydroxyphenyl group as an aromatic part. This
part was connected to a 4-carboxythiazolin-2-yl ring directly (an-
alog 1a), or was connected through one (analog 2a) or two (analog
2.1. Molecular design and synthesis of luciferin analogs
Because the scaffolds of these analogs are different from those of
existing substrates, we have used molecular modeling to predict
the interaction between intermediate species of one of the longest
analogs, 3b, and luciferase. Fig. 3 shows a plausible location of the
model compound oxy-3b and AMP at the active site of the
Photinus pyralis luciferase. The crystal structures used (PDB acces-
sion number 4G3623c and 2D1R32) were downloaded from the RSCB
protein data bank. Structural alignment, generation of electrostatic
surface potential and construction of the figure were carried out
using PyMOL (DeLano Scientific; http://www.pymol.org). The co-
ordinates of the DLSA-bound P. pyralis luciferase structure (chain B
of 3G36) superimposed closely on the oxyluciferin and AMP-bound
Luciola cruciata luciferase structure (2D1R), giving a root-mean-
3a) double bonds to study the effect of
p conjugation on the
emission wavelength (Fig. 2). A 4-(dimethylamino)phenyl group
was also selected as an aromatic part (1be3b) with an expectation
of a red shift of light emission, through the electron donating effect
of the alkylamino group. In addition, 6-hydroxynaphthalen-2-yl
analogs 1c23a and 2c were also anticipated as shifting emission
maxima toward red, because of longer p-conjugated systems than
ꢀ
square deviation of 1.1 A based on 3227 atoms. The local mini-
that of the corresponding 4-hydroxyphenyl analogs. The 3-
hydroxystyryl-type luciferin analog 2d was also prepared to eval-
uate the importance of the hydroxy group position for bio-
luminescence activity.
mum conformation model of oxy-3b was obtained using Spartan
’04 (Wavefunction; http://www.wavefun.com) by HartreeeFock 3-
21G calculation. The 4,5-dihydro-4-oxothiazol-2-yl moiety in oxy-
3b was manually aligned onto both a 4,5-dihydro-4-oxothiazol-2-yl
ring of the oxyluciferin and a thiazole ring of DLSA. As shown in
Fig. 3, the extended E,E-form of oxy-3b fitted into the narrow and
deep substrate-binding site. Several polar amino acid side chains
(Arg218, Ans229, Tyr255, Ser284, Glu311, and Arg337) faced the
The synthesis of luciferin analogs was conducted as shown in
Scheme 1. We utilized
ester for constructing the chiral thiazoline ring. Thus, the analogs
1a, 1b, 1c, and 2a were synthesized directly by the coupling of
cysteine with the corresponding nitriles, 4a, 4b, 4c, and 6,
D-cysteine or (S)-trityl-D-cysteine methyl
D
-

S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
3849
Scheme 1. Synthesis of aromatic analogs 1e3. Synthetic conditions: (a) D-Cys$HCl, NaOHaq, EtOH. (b) Acrylonitrile, Pd(OAc)2, CH3CO2K, K2CO3, water, reflux. (c) TBDMSCl, imid-
azole. (d) DIBAL. (e) Ph3P]CHCO2Et, toluene, reflux. (f) NaOHaq i-PrOH. (g) D-Cys(S-Trt)-OMe, EDC, DMAP, DMF, rt. (h) Ph3PO, Tf2O, CH2Cl2. (i) Esterase, EtOH, 10 mM NH4HCO3, pH
7.8, 36 ꢀC.
respectively. The analogs 2b, 2c, 2d, 3a, and 3b were obtained by
the enzymatic hydrolysis (porcine liver esterase) of the corre-
sponding methyl esters 11b, 11c, 11d, 15a, and 15b, which were
constructed from the corresponding carboxylic acids 10b, 10c, 10d,
2.2. Bioluminescence activity of luciferin analogs
Bioluminescence activity assays were performed with wild-type
P. pyralis luciferase and ATPeMg for each compound, and the
emitted light count was integrated for a fixed time (180 s). The
emission spectra were measured from 400 nm to 750 nm (Fig. 4).
14a, and 14b via coupling with (S)-trityl-
D-cysteine methyl ester
followed by the formation of the thiazoline rings.
Fig. 4. Bioluminescence emission spectra of compounds of (A) 4-hydroxyphenyl, (B) 4-(dimethylamino)phenyl, (C) 6-hydroxynaphthalen-2-yl analogs, and (D) natural substrate D-
LH2. All spectra were normalized at the emission maxima, and relative light emission intensity compared with that of natural D-LH2 is shown in parenthesis in %.

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S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
All the synthetic aromatic analogs except 2d exhibited light-
emitting activities. In preliminary measurements, we observed
double bond was used to estimate the UV wavelength of the organic
compounds.26 Even from this rough estimate, a þ100 nm shift per
double bond seemed to be a bigger shift than we expected.
that the light emission intensity of 1:1 mixtures of
approximately 1:3 those of pure -2b. This means that the in-
hibition ability of -2b and the optical purity of the analogs are not
D- and L-2b were
D
The other possible reason apart from p conjugation was sup-
L
posed to be a solvent effect. Previously, we have investigated the
light-color modulation mechanism using 5,5-dimethyloxyluciferin
and aminoluciferin analogs as model compounds, and showed
that it depended on the base/solvent combination.27,28 When the
emission species is exposed into the polar water from a generally
hydrophobic binding site, the emission color may shift to red.
Therefore, we presumed the environment of the emission species
crucial in evaluating the light emission ability of the analogs when
we use P. pyralis luciferase. Thus, we used the racemic mixtures
without further separation. The light emission intensity increased
proportionally with an increasing amount of luciferase and/or
ATPeMg (data not shown). Analogs 2b and 3b showed compara-
tively potent bioluminescence activity with 15% and 5% relative
light intensities compared with that of natural
However, the light emission intensities of the analogs other than 2b
and 3b were less than 1% of that of the natural substrate. Although
D
-LH2, respectively.
of the synthetic compounds was more polar than that for natural D-
LH2. The domain alteration hypothesis of firefly luciferase29,30
seems to be supportive of this assumption. Firefly luciferase is
a family of adenylating enzymes containing acyl- and aryl-CoA
synthetases, and nonribosomal peptide synthetases.31 Many of
them are proposed for the domain movement during the catalytic
reaction. According to their crystal structural studies,23c,32 the pu-
tative substrate-binding site of luciferase is within a cavity of the N-
terminal domain, and the AMP moiety is located in-between the N-
and C-terminal domains. When this domain movement occurred
the bioluminescent intensity of 3b is only 5% of that of D-LH2 in
aqueous solution, the absorption coefficient of blood against
675 nm light (lmax generated by bioluminescence of 3b) is less than
1:30 of that for 560 nm light (lmax generated by the bio-
luminescence of
bioluminescence of 3b passes through blood-containing tissue
better than that of -LH2 under the same conditions. Observed
emission maximum and light emission intensity for 1c (lmax
565 nm and 0.1% relative light yield compared with that of -LH2)
were considerably different from the previously reported values for
D-LH2). This means that the light generated by the
D
after the adenylation of D-LH2 and the C-terminal domain covered
the substrate inside the enzyme, the emission species was sup-
posed to exist in the hydrophobic environment. If we assumed that,
D
1c (lmax 524 nm, 1.5% relative light yield compared with that of
D
-
like native D-LH2, adenylated synthetic substrates did not induce
LH2 at pH 9.3).23a This discrepancy may be because of the difference
domain alteration, then the emission species might have been ex-
posed to water, which may have resulted in an emission shift to-
ward red. Study of the whole C-domain-lacking mutant of firefly
luciferase showed the importance of the C-terminal domain for
bioluminescence.33 Interestingly, this luciferase mutant, with an
altered N-domain only, retains its luminescence activity and
in the experimental conditions.
The inactive 3-hydroxystyryl-type luciferin analog 2d compared
with the active 4-hydroxystyryl-type luciferin analog 2a, as in the
case of phenolic hydroxy positional isomers of luciferin,24 suggested
thatsynthetic substrates bindandact ina similarmannertonative D-
LH2, and that the position of the hydroxyl group is one of the im-
portant factors forlightemission ability. However, the slowemission
profile indicated a difference of environment at the catalytic site of
the luciferase. The light emission from all the synthetic substrates
showed a slow rise to maximum intensity (about a minute), and was
showed red emission around 620 nm irrespective of pH with D-
LH2eAMP. We assumed this result also supports the presumption
that unnatural conformation of the C-domain causes a polar active
site environment for the synthetic compounds.
sustained for about 1 min, as distinct from lightemission from
which rapidly reached maximum intensity within a few seconds
followed by a steep decay (will be discussed below).
D
-LH2,
2.3. Bioluminescence activity of adenylated derivatives of
luciferin analogs
Among the bioluminescently active compounds, obvious struc-
tureeemission wavelength relationships were found. The insertion
of conjugated double bonds between an aromatic ring and a thia-
zoline ring showed an emission maximum wavelength with an
approximately 100 nm shift to red per double bond. In addition, the
emission maximum of 4-(dimethylamino)phenyl analogs (1be3b)
exhibited an approximately 30 nm shift to red, compared with
those of corresponding 4-hydroxyphenyl analogs (1ae3a). Simi-
larly, the emission maximum of 6-hydroxynaphthalen-2-yl analogs
(1c and 2c) exhibited an approximately 130 nm shift to red, com-
pared with those of the corresponding 4-hydroxyphenyl analogs
(1a and 2a). Thus, we obtained the synthetic luciferase substrates
emitting a variety of colors, such as blue/purple, blue, green/
yellow-green, and red. The shortest blue emission (lmax¼440 nm)
was observed for 1a. Notably, the longest wavelength observed for
3b (lmax¼675 nm) was in the NIR window region. Therefore, these
results represent the capability of designing light-emitting sub-
strates to obtain a wider range of emission wavelengths than those
Next, we compared the bioluminescence activity of D-LH2 and
analogs to the corresponding adenylated derivatives. The use of the
adenylated derivatives allows us to skip the adenylation step in
bioluminescence reaction (Fig. 1, Eq. 1), and allows the luciferase to
only catalyze the oxidation step of the adenylated substrates (Fig. 1,
Eq. 2). If the adenylation step is the rate-determining step in light
emission, the speed of light emission should be increased to en-
hance light emission intensity. The adenylated derivatives were
prepared by reported procedures,34 purified by HPLC just prior to
use, and subjected to bioluminescence reaction. The adenylated
substrate derivatives were rather stable under acidic conditions
and no epimerization or hydrolysis was observed in pH 4 buffer
solution or in 0.05% TFA-containing HPLC eluent over 1 h at room
temperature. However, they were somewhat unstable under basic
conditions measuring bioluminescence activity: the half-life for
hydrolysis and epimerization in the pH 8 buffer was ca. 1 h and
10 min, respectively, at room temperature. Thus, the adenylated
material was prepared and purified by HPLC under acidic condi-
tions just prior to use. The bioluminescence emission profiles of the
most active anaolg 2b and its adenylated 2beAMP are shown in
Fig. 5 as representative results. The emission maxima were not
changed by adenylation, though the emission intensity was en-
hanced 10 to several hundred times with shorter rise times (8 s), as
from native
One of the reasons for thesewavelengthshifts was indicated tobe
that the length of the conjugation controls the wavelength of the
D-LH2.
p
emission maximum. Theoretical calculation for the excited species
and prediction of the spectroscopic properties are challenging
fields,25 especially the characterization of light emitters (excited
oxyluciferins) interacting with various intramolecular factors, in-
cluding hydrogen bonds and Coulomb interaction, and polarity in
the luciferase active site. Commonly, a þ30 nm shift per conjugated
for D-LH2 (6 s). Thus, the slow rise of light emission intensity of the
analog itself was suggested to be responsible for the slow reaction
of the adenylation step. The emission intensity of adenylated ana-
logs decayed more slowly than that of adenylated D-LH2. Compared

S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
3851
Fig. 5. Comparison of changes in bioluminescence over time of 2b/ATP with 2beAMP, and of D-LH2/ATP with D-LH2eAMP. The most active 2b was selected as a delegate for
synthesized compounds. (A) 2b/ATP (thick line) and 2beAMP (dashed line). (B) D-LH2/ATP (thick line) and D-LH2eAMP (dashed line). Relative light emission intensity of 2beAMP
compared with D-LH2eAMP is shown in parenthesis in %.
with
D-LH2,
D
-LH2eAMP showed a burst of light emission followed
without further purification. Porcine liver esterase was purchased
by drastic decay. If this quick decay of
D
-LH2eAMP is because of
from Sigma (E3019, lyophilized powder). Solvents used for anhy-
drous conditions were distilled, or dried over 4 A molecular sieves.
product inhibition,1 synthetic analogs seem to have fewer product
ꢀ
inhibition properties compared with those of oxyluciferin from
natural D-LH2. The synthetic analogs for P. pyralis luciferase may be
useful for in vivo whole-body imaging, because of the duration of
substantial maximum light production suitable for detecting more
stable signals, coupled with availability of vectors for luciferase
gene to introduce it into living organisms.
Recently, bioluminescence resonance energy transfer (BRET)
systems utilizing chemically modified liciferin18,19 and firefly lu-
ciferase17,35 were successfully developed to obtain light emission in
the NIR window region,18,19 and the longest wavelength was
recorded at 783 nm.35 This is a far longer wavelength than we
observed from 3b, though our compounds do not require the in-
jection of modified luciferase into organisms to obtain red emis-
sion. Nevertheless, our compounds may also have the potential to
be used as BRET donors. It should be noted that according to the
functional specification of the photomultiplier tube (Hamamatsu
R4220) of the luminometer used, sensitivity at 700 nm is more than
10 times less than at 600 nm. We will therefore be required to
prepare more suitable apparatus for further development of lucif-
erase substrates with NIR range emission.
Cation exchange resins (Organo, Amberlite IR-120B NA, and
IRA400OH AG) were used to remove ions. Merck precoated Kie-
selgel 60 F254 plates with 0.25 mm (Art. 5715) and 0.5 mm (Art.
5744) thickness were used for analytical and preparative thin layer
chromatography (TLC). Visualization of TLC was accomplished with
UV-light and by treatment with suitable staining reagents. Merck
Kieselgel 60 (Art. 7734) was used for column chromatography.
Solvents were removed with a rotary evaporator under reduced
pressure at a temperature below 40 ꢀC. Melting points were mea-
sured using a Yamato MP-2 instrument and are uncorrected. Op-
tical purity of synthesized luciferin analogs was analyzed by HPLC
(Agilent 1100 series) using a chiral column (Daicel Chemical In-
dustries, OD-RH or OZ-RH, 5
m
m, 4.6ꢁ150 mm) with linear gradient
of 10%e90% acetonitrile in H2O over 30 min (flow rate 0.5 mL/min)
as eluent. Unless otherwise stated, chiral analyses were performed
using the OD-RH column. A UV detector set at 330 nm was used for
peak detection. The retention times of L-LH2 and D-LH2 were 17.2
and 18.2 min, respectively. Synthesized compounds were used for
the luminescence assay without further purification. IR spectra
were measured using a Horiba FT 730 spectrometer. 1H and 13C
NMR spectra were recorded on JEOL Lambda 270 [270 MHz (1H)
and 67.8 MHz (13C)] and JEOL ECA 500 [500 MHz (1H) and 125 MHz
3. Conclusion
(
(
13C)] instruments. Chemical shifts are reported in parts per million
We developed simple luciferin analogs with various light
emission colors ranging from blue to the NIR window region. From
study of the structureeemission wavelength relationship, we found
that the introduction of conjugated double bonds between aro-
matic parts and the thiazoline ring was very effective for elongation
of wavelengths of emission maxima, producing an approximately
100 nm longer wavelength shift per conjugated double bond. The
shortest emission wavelength was observed with 1a (lmax 440 nm),
and the longest with 3b (lmax 675 nm), reached within the NIR
window region of light suitable for bioimaging of deep sites in
living animals. The light emission from analogs showed slow in-
creases to maximum intensity (about a minute), and was sustained
d) downfield from internal tetramethylsilane (d¼0) and coupling
constants in hertz. Fast atom bombardment mass spectra (FAB-MS)
were measured with a Finnigan MAT TSQ-700 instrument (glycerol
matrix). Electron ionization mass spectra (EI-MS) were measured
with a JEOL JMS 600H instrument. High-resolution electrospray
ionization mass spectra (HR-ESI-MS) were measured with JEOL JMS
T1000LC mass spectrometers, using tuned conditions of needle
voltage: 2000 V, orifice 1 voltage: 85 V, orifice 2 voltage: 5 V, ring
lens voltage: 10 V, desolvating gas: 250 ꢀC, orifice 1 temperature:
80 ꢀC, delivery of sample: infusion method, flow speed: 10e30
mL/
min (according to the sample).
for about 1 min, being different from that of
D
-LH2 light emission,
4.1.2. Materials and general method for luminescence assay. A 1 mg/
mL stock solution of commercial luciferase purchased from Prom-
ega (QuantiLum recombinant P. pyralis luciferase, E1701) and Sigma
(L9506) was prepared by dissolving the luciferase in 50 mM
TriseHCl buffer (pH 8.0) containing 10% glycerol and was stored at
ꢂ80 ꢀC. Just prior to use, the luciferase stock solutions were diluted
100-fold with 50 mM potassium phosphate buffer (pH 6.0) con-
taining 35% glycerol. The diluted luciferase solution was ice-cooled
until use. ATPeMg was purchased from Nacalai Tesque, and the
buffer chemicals from Wako Chemicals or Kanto Chemicals. Stock
solution of substrates was prepared by dissolving 5 mM of the
substrates in 50 mM potassium phosphate buffer (pH 6.0), and was
which rapidly reaches maximum intensity within a few seconds
followed by a steep decay. The rather weak bioluminescence in-
tensity of analogs could be enhanced 10 to several hundred times
by their derivatization to corresponding adenylated substrates.
4. Experimental section
4.1. General
4.1.1. Materials and general method for synthesis. Starting materials
and reagents were obtained from commercial suppliers and used

3852
S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
stored at ꢂ80 ꢀC. Deionized water (Millipore, Milli-RX-12
a) was
preparation of 1b, 6-cyano-2-naphthol (4c) (102 mg, 0.60 mmol)
was treated with -Cys$HCl (290 mg, 1.65 mmol) and 1 M NaOH
used for aqueous assay solutions. pH was monitored with a Horiba
F-23 pH meter. Bioluminescence intensity was measured using an
ATTO AB-2200 or AB-2270 luminometer (Hamamatsu, R4220
photomultiplier tube) and bioluminescence spectra were recorded
using an ATTO AB-1850 spectrophotometer. Adenylated derivatives
were prepared according to the literature,34 and purified using an
HPLC system (Agilent 1100 series) equipped with a Mightysil C18
D
(2.5 mL) in EtOH (5 mL) to afford anaolg 1c quantitatively as
a yellow solid. Mp 197 ꢀC dec (lit.23a 201.5e203.5 ꢀC dec); 90% ee
from chiral HPLC (retention time of
L
-isomer: 10.8 min,
D-iso-
mer: 11.2 min); 1H NMR (270 MHz, CD3OD)
d
3.71 (dd, J¼8.9,
11.2 Hz, 1H, ABX system), 3.76 (dd, J¼8.9, 11.2 Hz, 1H, ABX sys-
tem), 5.33 (dd, J¼8.9, 8.9 Hz, 1H, ABX system), 7.12e7.16 (com-
plex, 2H), 7.68 (d, J¼8.9 Hz, 1H), 7.83 (d, J¼9.6 Hz, 1H), 7.90 (dd,
J¼1.6, 8.9 Hz, 1H), 8.21 (br d, J¼1.6 Hz, 1H); 13C NMR (67.8 MHz,
reverse phase column (5
m
m, 4.6ꢁ250 mm) just prior to use. The
flow rate of the mobile phase consisting of a mixture of acetonitrile
and 0.05% TFA aqueous solution was 0.5 mL/min, and the column
temperature was maintained at 20 ꢀC. A linear gradient of 10%e90%
acetonitrile over 30 min was applied, and a UV detector set at
CD3OD)
d 37.1 (t), 83.0 (d), 110.0 (d), 120.3 (d), 126.2 (d), 127.4
(d), 128.8 (s), 128.9 (s), 130.4 (d), 131.7 (d), 138.1 (s)ꢁ2, 158.4 (s),
170.0 (s), 178.5 (s); FT-IR nmax (cmꢂ1): 3022, 1589, 1483; HR-ESI-
MS m/z: [MþH]þ calcd for C14H12NO3S, 274.0538; found,
247.0533, and [MþHꢂCO2]þ calcd for C13H12NOS, 230.0640;
found, 230.0644.
330 nm was used for peak detection.
13.4 min, followed by -LH2eAMP (13.6 min), and
-2beAMP was eluted at 22.2 min, followed by
D
-LH2eAMP was eluted at
-LH2 (20.9 min).
-2beAMP
L
D
D
L
(22.8 min), and 2b (26.8 min). The volatiles of the eluted fraction
were removed under reduced pressure and the residual aqueous
solution containing adenylated product was promptly subjected to
bioluminescence activity. The purity and concentration of the
synthesized adenylates were determined to be more than 95% us-
ing a calibration curve prepared with the HPLC system.
4.2.4. (S,E)-2-(4-Hydroxystyryl)-4,5-dihydrothiazole-4-carboxylic
acid (2a). Following the literature,36 to a solution of 4-iodophenol
(5) (1100 mg, 5.0 mmol) in water (10 mL) were added CH3CO2K
(491 mg, 5.0 mmol), K2CO3 (865 mg, 6.3 mmol), acrylonitrile
(495
mL, 7.5 mmol), and Pd(OAc)2 (11 mg, 0.05 mmol), and the
mixture was heated under reflux for 30 min. After the reaction
mixture was cooled to room temperature, precipitates were filtered
off through a pad of Celite and the filter cake was washed with
water (50 mL). The filtrate and washings were combined, adjusted
to pH 7 with 2 M HCl, and the products were extracted with EtOAc
(40 mL). The organic layers were combined, dried over Na2SO4, and
concentrated to leave a white solid. Recrystallization from EtOAc/i-
Pr2O gave nitrile 6 (417 mg, 58%) as colorless needles being a mix-
ture of stereoisomers (cis/trans¼1:3 by 1H NMR). 1H NMR
4.2. Preparation of luciferin analogs
4.2.1. (S)-2-(4-Hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic
acid (1a). To a solution of 4-cyanophenol (4a) (120 mg, 1.01 mmol)
and
1.20 mmol) in EtOH (5 mL) was added 1 M NaOH (5 mL). After the
reaction mixture was stirred at 80 ꢀC for 6 h, additional
-Cys$HCl
D-cysteine hydrochloride monohydrate (D-Cys$HCl) (211 mg,
D
(422 mg, 2.40 mmol) and 1 M NaOH (1 mL) were added, and the
mixture was stirred at room temperature for 4 days. The reaction
mixture was filtered and the filtration residue was washed with
50% aqueous EtOH (20 mL). The filtrate and washings were com-
bined and the pH of the mixture was adjusted to pH 2 with 2 M HCl.
The precipitates were collected by suction filtration, washed by
distilled water, and dried under reduced pressure to give anaolg 1a
(111 mg, 50%) as a white powder. Mp 200e204 ꢀC dec; 90% ee from
(500 MHz, CDCl3)
d
5.30 (d, J¼12.5 Hz, 0.25H cis), 5.85 (d, J¼16 Hz,
0.75H trans), 6.75 (d, J¼8 Hz, 1.5H trans, AA0BB0 system), 6.77 (d,
J¼9.0 Hz, 0.5H cis, AA0BB0 system), 7.08 (d, J¼12.5 Hz, 0.25H cis),
7.33 (d, J¼16 Hz, 0.75H trans), 7.35 (d, J¼8 Hz, 1.5H trans, AA0BB0
system), 7.66 (d, J¼9.0 Hz, 0.5H cis, AA0BB0 system). To a solution of
D-Cys$HCl (106 mg, 0.60 mmol) and nitrile 6 (65.0 mg, 0.45 mmol)
in MeOH (2 mL) was added 1 M NaOH (2 mL) and the mixture was
stirred at 80 ꢀC for 6 h. The reaction mixture was neutralized with
1 M HCl and was directly purified using a Sep-Pak cartridge (Wa-
ters, C18, water to 60% MeOH stepwise gradient) to give anaolg 2a
(9.2 mg, 8%) as a pale-yellow solid. Mp 138e140 ꢀC dec; 98% ee;
chiral HPLC (retention time of
L
-isomer: 8.0 min,
d
D-isomer:
3.70 (dd, J¼7.9, 11.5 Hz, 1H,
8.7 min); 1H NMR (270 MHz, CD3OD)
ABX system), 3.76 (dd, J¼8.9, 11.5 Hz, 1H, ABX system), 5.23 (dd,
J¼7.9, 8.9 Hz, 1H, ABX system), 6.85 (d, J¼8.9 Hz, 2H, AA0BB0 sys-
tem), 7.74 (d, J¼8.9 Hz, 2H, AA0BB0 system); 13C NMR (67.8 MHz,
from chiral HPLC (retention time of
L
-isomer: 12.3 min,
D-isomer:
CDCl3)
d
36.0 (t), 78.0 (d), 116.5 (d)ꢁ2, 124.5 (s), 131.8 (d)ꢁ2, 163.0
12.9 min); 1H NMR (270 MHz, CD3OD)
d
3.52 (dd, J¼8.9, 10.9 Hz, 1H,
(s), 173.9 (s), 174.6 (s); FT-IR nmax (cmꢂ1): 2680, 1610, 1580, 1510; EI-
MS m/z: 223 (Mþ, 44%), 178 (100). HR-ESI-MS: m/z: [MþNa]þ calcd
for C10H9NNaO3S, 246.0201; found, 246.0157.
ABX system), 3.61 (dd, J¼8.9, 10.9 Hz, 1H, ABX system), 5.01 (dd,
J¼8.9, 8.9 Hz, 1H, ABX system), 6.80 (d, J¼8.9 Hz, 2H, AA0BB0 sys-
tem), 6.91 (d, J¼16.0 Hz, 1H), 7.10 (d, J¼16.0 Hz, 1H), 7.42 (d,
J¼8.9 Hz, 2H, AA0BB0 system); 13C NMR (67.8 MHz, CDCl3)
d 36.5 (t),
4.2.2. (S)-2-(4-(Dimethylamino)phenyl)-4,5-dihydrothiazole-4-
carboxylic acid (1b). To a solution of 4-(dimethylamino)benzoni-
trile (4b) (103 mg, 0.71 mmol) and D-Cys$HCl (371 mg, 2.12 mmol)
in EtOH (4 mL) was added 1 M NaOH (5 mL). After the reaction
mixture was stirred at 80 ꢀC for 5 h, 1 M HCl (5 mL) was added, and
the mixture was concentrated under reduced pressure. The residue
was washed with distilled water to give analog 1b (50.9 mg, 29%) as
81.2 (d), 116.9 (d)ꢁ2,119.7 (d), 128.0 (s), 130.5 (d)ꢁ2,143.7 (d),160.7
(s), 172.0 (s), 177.5 (s); FT-IR nmax (cmꢂ1): 3151, 1626, 1568; ESI-MS
m/z: 250 [(MþH)þ]. HR-ESI-MS: m/z: [MþH]þ calcd for C12H12NO3S,
250.0538; found, 250.0516.
4.2.5. S-Trityl-
D-cysteine methyl ester [
D-Cys(S-Trt)-OMe]. To a solu-
tion of S-trityl-
D
-cysteine (504 mg, 1.39 mmol) in MeOH (100 mL)
a yellow solid. 92% ee from chiral HPLC (retention time of
L
-isomer:
was added 4 M HCl (5.4 mL in 1,4-dioxane), and the mixture was
stirred at ambient temperature for 17 days. The reaction mixture
was neutralized by adding ion exchange resin IRA400OH AG. The
resin was filtered off and washed with MeOH. The filtrate and
washings were combined and concentrated in vacuo. The residue
was purified by silica gel column chromatography (Hex/
11.1 min,
D
-isomer: 11.5 min); 1H NMR (270 MHz, CDCl3)
d 3.01 (s,
6H), 3.50 (dd, J¼9.2, 11 Hz, 1H, ABX system), 3.61 (dd, J¼9.2, 11 Hz,
1H, ABX system), 5.00 (dd, J¼9.2, 9.2 Hz, 1H, ABX system), 6.71 (d,
J¼7.0 Hz, 2H, AA0BB0 system), 7.71 (d, J¼7.0 Hz, 2H, AA0BB0 system);
FT-IR nmax (cmꢂ1): 3392, 1608; ESI-MS m/z: 251 [(MþH)þ]. HR-ESI-
MS: m/z: [MþNa]þ calcd for C12H14N2NaO2S, 273.0674; found,
273.0649.
EtOAc¼1:1) to give
D-Cys(S-Trt)-OMe 455 mg (86%) as a pale-
yellow oil. 1H NMR (270 MHz, CDCl3)
d
2.47 (dd, J¼7.7, 12.4 Hz,
1H, ABX system), 2.60 (dd, J¼4.8, 12.4 Hz, 1H, ABX system), 3.20
(br dd, J¼4.8, 7.7 Hz, 1H, ABX system), 3.65 (s, 3H), 7.18e7.31
(complex, 9H, 3ꢁ C6H3), 7.40e7.45 (complex, 6H, 3ꢁ C6H2); 13C
4.2.3. (S)-2-(6-Hydroxynaphthalen-2-yl)-4,5-dihydrothiazole-4-
carboxylic acid (1c). In a similar manner to that used for the

S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
3853
NMR (67.8 MHz, CDCl3)
d
36.9 (t), 52.1 (q), 53.8 (d), 66.8 (s), 126.8
chromatography (Hex/EtOAc¼8:1) to yield 6-(tert-butyldime-
thylsilyloxy)naphthalene-2-carbonitrile (7) (69.9 mg, 83%) as
(d)ꢁ3, 127.9 (d)ꢁ6, 129.6 (d)ꢁ6, 144.5 (s)ꢁ3, 174.2 (s); FT-IR nmax
(cmꢂ1): 3381, 3315, 1739, 1595; FAB-MS m/z: 378 (MþHþ, 10%),
243 (100).
a colorless oil. 1H NMR (270 MHz, CD3OD)
d 0.23 (s, 6H), 0.97 (s, 9H),
7.12 (complex, 2H), 7.48 (dd, J¼1.6, 8.6 Hz, 1H), 7.69 (d, J¼8.9 Hz,
1H), 7.73 (d, J¼8.9 Hz, 1H), 8.08 (d, J¼0.5 Hz, 1H); FT-IR nmax (cmꢂ1):
3689, 2225, 1274; ESI-MS m/z: 284 [(MþH)þ], 306[(MþNa)þ]. Ni-
trile 7 (99.3 mg, 0.35 mmol) was dissolved in toluene (10 mL) under
Ar. To the mixture was added 1 M solution of diisobutylaluminium
hydride (DIBAL) in toluene solution (0.5 mL). After the reaction
mixture was stirred at room temperature for 1 h, acetone (10 mL)
was added to the ice-water-cooled reaction mixture for de-
composition of the excess reagent. To the mixture were added satd
potassium sodium tartrate (20 mL) and water (30 mL). The prod-
ucts were extracted from the suspension with EtOAc (3ꢁ50 mL).
The combined organic layers were dried over Na2SO4, and evapo-
rated. The residue was purified by PTLC (Hex/EtOAc¼10:1) to afford
aldehyde 8 (74.4 mg, 74%) as a yellow oil. 1H NMR (270 MHz, CDCl3)
4.2.6. (S,E)-2-(4-(Dimethylamino)styryl)-4,5-dihydrothiazole-4-
carboxylic acid (2b). To a solution of 4-(dimethylamino)cinnamic
acid (10b) (92.1 mg, 0.48 mmol) in DMF (5 mL) were added
Cys(S-Trt)-OMe (101 mg, 0.53 mmol), 1-ethyl-3-(3-
dimethylamino-propyl)carbodiimide (EDC) (311 mg, 1.62 mmol),
and 4-(dimethylamino)pyridine (DMAP) (151 mg, 1.23 mmol)
under Ar. After the reaction mixture was stirred at room tem-
perature for 24 h, water (100 mL) was added. The products were
extracted with EtOAc (3ꢁ100 mL). The combined organic layers
were dried over Na2SO4, filtered, and concentrated. The residue
obtained was purified by silica gel column chromatography (Hex/
D-
EtOAc¼1:1) to give an N-acyl-S-trityl
D-cysteine methyl ester de-
rivative, (S)-methyl 2-((E)-3-(4-(dimethylamino)phenyl)acryl-
d
0.28 (s, 6H), 1.03 (s, 9H), 7.17 (dd, J¼2.3, 8.6 Hz, 1H), 7.23 (br d,
amido)-3-(tritylthio)propanoate (259 mg, 89%) as a yellow oil
J¼2.3 Hz, 1H), 7.76 (d, J¼8.6 Hz, 1H), 7.89 (d, J¼8.6 Hz, 1H), 7.90 (dd,
J¼1.6, 8.6 Hz, 1H), 8.26 (s, 1H), 10.09 (s, 1H); FT-IR nmax (cmꢂ1): 1716,
1274; ESI-MS m/z: 287 [(MþH)þ]. To a solution of aldehyde 8
(63.9 mg, 0.22 mmol) in toluene (2 mL) was added
ethoxycarbonylmethylene-triphenylphosphoran (Ph3P]CHCO2Et)
(121 mg, 0.349 mmol) and the mixture was stirred at room tem-
perature for 5 h. After dilution of the reaction mixture with water
(50 mL), the products were extracted with EtOAc (3ꢁ50 mL). The
combined organic layers were dried over Na2SO4 and evaporated.
The residue was purified by PTLC (Hex/EtOAc¼25:1) to give ester 9
(76.6 mg, 97%) as a yellow oil (step e in Scheme 1). 1H NMR
(step g in Scheme 1). 1H NMR (500 MHz, CDCl3)
d 2.72 (m, 2H, ABX
system), 3.01 (s, 6H), 3.72 (s, 3H), 4.78 (m, 1H, ABX system), 6.15
(d, J¼15.5 Hz, 1H), 6.20 (d, J¼5.9 Hz, 1H, NH), 6.68 (d, J¼8.6 Hz, 2H,
AA0BB0 system), 7.20e7.42 (complex, 17H), 7.52 (d, J¼15.5 Hz, 1H);
ESI-MS m/z: 573 [(MþNa) þ]. To a solution of the amide obtained
above (118 mg, 0.21 mmol) in dry CH2Cl2 (10 mL) were added
triphenylphosphine oxide (Ph3PO) (124 mg, 0.45 mmol) and tri-
fluoromethanesulfonic anhydride (Tf2O) (360 mL, 2.14 mmol) un-
der Ar.37 After the reaction mixture was stirred at room
temperature for 40 min, the reaction was quenched by adding
water (50 mL), and the products were extracted with CHCl3
(50 mL), and then EtOAc (2ꢁ50 mL). The combined organic layers
were dried over Na2SO4, filtered, and concentrated. The residue
was purified by silica gel column chromatography (Hex/
EtOAc¼1:2) to give thiazoline ester 11b (44.2 mg, 71%) as a yellow
(270 MHz, CDCl3)
d
0.25 (s, 6H), 1.00 (s, 9H), 1.34 (t, J¼7.0 Hz, 3H),
4.27 (q, J¼7.0 Hz, 2H), 6.47 (d, J¼16.1 Hz, 1H), 7.11 (dd, J¼2.4, 8.9 Hz,
1H), 7.19 (d, J¼2.4 Hz, 1H), 7.60e7.79, complex, 4H), 7.85 (br s, 1H);
FT-IR nmax (cmꢂ1): 1623, 1274; ESI-MS m/z: 357 [(MþH)þ]. To a so-
lution of ester 9 (90.8 mg, 0.253 mmol) in i-PrOH (3 mL) was added
1 M NaOH (5 mL) and the mixture was stirred at room temperature
for 5 h. The reaction mixture was neutralized by adding ion ex-
change resin IR-120B NA. The resin was removed by filtering. The
filtrate and washings were combined, and evaporated to give acid
10c quantitatively as a pale-yellow oil (step f in Scheme 1). 1H NMR
solid (step h in Scheme 1). 1H NMR (500 MHz, CDCl3)
d 2.96 (s,
6H), 3.53 (m, 2H, ABX system), 3.79 (s, 3H), 5.14 (dd, J¼8.6, 8.6 Hz,
1H, ABX system), 6.63 (d, J¼7.5 Hz, 2H, AA0BB0 system), 6.88 (d,
J¼15.5 Hz, 1H), 7.04 (d, J¼15.5 Hz, 1H), 7.34 (d, J¼7.5 Hz, 2H, AA0BB0
system); FT-IR nmax (cmꢂ1): 1724; ESI-MS m/z: 291 [(MþH)þ]. To
a mixture of thiazoline ester 11b (21.1 mg, 0.07 mmol) in EtOH
(2 mL) and 10 mM NH4HCO3 (6 mL) was added porcine liver es-
terase (9.2 mg), and the reaction mixture was stirred at 37 ꢀC
under Ar for 19 h. After evaporation of the reaction mixture, the
residue obtained was suspended in a mixture of MeOH/CHCl3. The
precipitate was filtered off, and the filtrate and washings of the
residue were combined and concentrated to give anaolg 2b
(14.1 mg, 71%) as an orange solid (step i in Scheme 1). 26% ee from
(270 MHz, CD3OD)
d
6.50 (d, J¼15.7 Hz, 1H), 7.10 (dd, J¼2.2, 7.6 Hz,
1H), 7.11 (br s, 1H), 7.65 (m, 2H), 7.77 (dd, J¼1.6, 8.1 Hz, 1H), 7.80 (d,
J¼15.7 Hz, 1H), 7.90 (s 1H); FT-IR nmax (cmꢂ1): 3689, 1670; ESI-MS
m/z: 237 [(MþNa)þ]. In a similar manner to that used in step g in
the synthesis of analog 2b, acid 10c (54.9 mg, 0.25 mmol)
was coupled with
-cysteine methyl ester derivative, (S)-methyl 2-((E)-3-
(2-hydroxynaphthalen-6-yl)acrylamido)-3-(tritylthio)propanoate
(58.4 mg, 40%) as a pale-yellow oil. 1H NMR (270 MHz, CDCl3)
2.75
D-Cys(S-Trt)-OMe to give an N-acyl-S-trityl
D
chiral HPLC (retention time of
L-isomer: 13.2 min,
D
-isomer:
d
12.9 min); 1H NMR (500 MHz, CD3OD)
d
3.00 (s, 6H), 3.62 (m, 2H,
(m, 2H, ABX system), 3.75 (s, 3H), 4.77 (dd, J¼2.7, 7.9 Hz, 1H, ABX
system), 6.35 (d, J¼16.1 Hz, 1H), 6.90e7.80 (complex, 23H); FT-IR
nmax (cmꢂ1): 3689, 3565, 1868; ESI-MS m/z: 574 [(MþH) þ]. In
a similar manner to that used in step h in the synthesis of analog 2b,
the amide obtained above (60.3 mg, 0.11 mmol) was cyclized to
yield thiazoline ester 11c (17.4 mg, 55%) as a yellow solid. 1H NMR
ABX system), 5.01 (dd, J¼8.6, 8.6 Hz, 1H, ABX system), 6.72 (d,
J¼9.0 Hz, 2H, AA0BB0 system), 6.86 (d, J¼16 Hz, 1H) 7.21 (d,
J¼16 Hz, 1H), 7.44 (d, J¼9.0 Hz, 2H, AA0BB0 system); 13C NMR
(125 MHz, CD3OD,
d
): 31.2 (t), 34.5 (q)ꢁ2, 79.7 (d), 112.5 (d)ꢁ2,
115.9 (d), 122.5 (s), 130.1 (d)ꢁ2, 144.1 (d), 152.1 (s), 170.4 (s), 172.2
(s); FT-IR nmax (cmꢂ1): 3392, 1602; HR-ESI-MS m/z: [MþH]þ calcd
for C14H17N2O2S, 277.1011; found, 277.0989.
(270 MHz, CDCl3) d 3.63 (m, 2H, ABX system), 3.81 (s, 3H), 5.27 (dd,
J¼8.9, 8.9 Hz, 1H, ABX system), 7.07e7.13 (complex, 3H), 7.33 (d,
J¼16.1 Hz, 1H), 7.65 (complex, 2H), 7.76 (m, 1H), 7.88 (br s, 1H); FT-
IR nmax (cmꢂ1): 3689, 1733; ESI-MS m/z: 314 [(MþH)þ], 336
[(MþNa)þ]. In a similar manner to that used in step i in the syn-
thesis of analog 2b, thiazoline ester 11c (6.3 mg, 0.02 mmol) was
hydrolyzed with porcine liver esterase to give anaolg 2c quantita-
tively as a pale-yellow solid. 22% ee from chiral HPLC (OZ-RH col-
4.2.7. (S,E)-2-(2-(6-Hydroxynaphthalen-2-yl)vinyl)-4,5-dihydrothi-
azole-4-carboxylic acid (2c). To a solution of 6-cyano-2-naphthol
(4c) (50.2 mg, 0.30 mmol) in DMF (0.5 mL) were added tert-
butyldimethylsilyl chloride (TBDMSCl, 143 mg, 0.95 mmol) and
imidazole (160.7 mg, 2.40 mmol). After the mixture was stirred at
room temperature for 1 h, water (40 mL) was added. The products
were extracted from the diluted mixture with EtOAc (3ꢁ60 mL).
The combined organic layers were dried over Na2SO4, filtered, and
concentrated. The residue was purified by silica gel column
umn, retention time of
NMR (270 MHz, CD3OD)
8.9 Hz, 1H, ABX system), 7.06e7.17 (complex, 3H), 7.30 (dd,
J¼16.1 Hz, 1H), 7.65e7.87 (complex, 4H); FT-IR nmax (cmꢂ1): 3689,
L-isomer: 17.4 min, D
-isomer: 18.2 min; 1H
d
3.61 (m, 2H, ABX system), 5.09 (dd, J¼8.9,

3854
S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
1716; ESI-MS m/z: 300 [(MþH)þ]. HR-ESI-MS m/z: [MþH]þ calcd for
residue was purified by PTLC (Hex/EtOAc¼2:1) to obtain 4-(tert-
C16H14NO3S, 300.0694; found, 300.0689.
butyldimethylsilyloxy)cinnamyl alcohol (141 mg, 83%) as a color-
less oil. 1H NMR (500 MHz, CDCl3)
d 0.21 (s, 6H), 0.98 (s, 9H), 4.30
4.2.8. (S,E)-2-(3-Hydroystyryl)-4,5-dihydrothiazole-4-carboxylic
(br s, 2H), 6.25 (d, J¼16 Hz, 1H), 6.56 (d, J¼16 Hz, 1H), 6.80 (d,
J¼8.6 Hz, 2H, AA0BB0 system), 7.28 (d, J¼8.6 Hz, 2H, AA0BB0 system);
FT-IR nmax (cmꢂ1): 3388; EI-MS m/z: 264 (Mþ, 54%), 207 (100). To
the 4-(tert-butyldimethylsilyloxy)cinnamyl alcohol obtained above
(233 mg, 0.88 mmol) in CH2Cl2 (80 mL) was added MnO2 (1.40 g,
16.1 mmol) and the mixture was vigorously stirred at room tem-
perature for 4 h. The reaction mixture was filtered through a pad of
Celite and the filtration cake was washed thoroughly with CH2Cl2.
The filtrate and washings were combined and evaporated to give 4-
(tert-butyldimethylsilyloxy)-cinnamaldehyde (12a) (229 mg, 99%)
as a pale-yellow oil. A small portion was purified by PTLC (Hex/
EtOAc¼2:1) for analysis, and the rest was used for the next reaction
acid (2d). To
a solution of 3-hydroxycinnamic acid (1.80 g,
11.0 mmol) in CH2Cl2 (200 mL) were added acetic anhydride
(4.0 mL, 42 mmol) and DMAP (6.70 g, 54.8 mmol), and the mixture
was stirred at room temperature for 4 h. Water (150 mL) was added
to the reaction mixture and the products were extracted from the
diluted mixture with CH2Cl2 (100 mL) and EtOAc (2ꢁ80 mL) suc-
cessively. The organic layers were combined, dried over MgSO4, and
concentrated. The residue was purified by silica gel column chro-
matography (Hex/EtOAc¼2:1) to yield 3-acetoxycinnamic acid
(10d) (1.28 g, 71%) as colorless needles. Mp 140e142 ꢀC; 1H NMR
(270 MHz, CD3OD)
d
2.28 (s, 3H), 6.49 (d, J¼16.0 Hz, 1H), 7.12 (d,
J¼7.4 Hz, 1H, AA0BB0 system), 7.34e7.46 (complex, 3H), 7.63 (d,
step without further purification. 1H NMR (270 MHz, CDCl3)
d 0.22
J¼16.0 Hz, 1H); 13C NMR (67.8 MHz, CD3OD)
d
20.9 (q), 121.0 (d),
(s, 6H), 0.98 (s, 9H), 6.58 (dd, J¼7.8, 16 Hz, 1H), 6.87 (d, J¼8.6 Hz, 2H,
AA0BB0 system), 7.41 (d, J¼16 Hz, 1H), 7.46 (d, J¼8.6 Hz, 2H, AA0BB0
system), 9.64 (d, J¼7.8 Hz, 1H); FT-IR nmax (cmꢂ1): 1675; EI-MS m/z:
262 (Mþ, 47%), 207 (25), 206 (100). In a similar manner to that used
in step e in the synthesis of analog 2c, aldehyde 12a (47.6 mg,
0.18 mmol) was subjected to Wittig olefination to give ester 13a
122.1 (d), 124.6 (d), 126.6 (d), 130.9 (d), 137.5 (s), 144.8 (s), 152.7 (s),
170.5 (s), 171.0 (s); FT-IR nmax (cmꢂ1): 3037, 1761, 1687, 1631; EI-MS
m/z: 206 (Mþ, 26%), 164 (100). In a similar manner to that used in
step g in the synthesis of analog 2b, acid 10d (133 mg, 0.65 mmol)
was coupled with D-Cys(S-Trt)-OMe to give an N-acyl-S-trityl D-
cysteine methyl ester derivative, (S)-methyl 2-((E)-3-(3-
(45.4 mg, 76%) as a yellow oil. 1H NMR (270 MHz, CDCl3)
d 0.22 (s,
acetoxyphenyl)acrylamido)-3-(tritylthio)propanoate (159 mg,
6H), 0.99 (s, 9H), 1.32 (t, J¼7.3 Hz, 3H), 4.25 (q, J¼7.3 Hz, 2H), 6.00
(dd, J¼7.8, 15 Hz, 1H), 6.76e6.89 (complex, 4H), 7.36 (d, J¼7 Hz, 2H),
7.46 (d, J¼15 Hz, 1H); FT-IR nmax (cmꢂ1): 1705; EI-MS m/z: 332 (Mþ,
41%), 275 (22), 218 (100). In a similar manner to that used in step f
in the synthesis of analog 2c, ester 13a (9.7 mg, 0.03 mmol) was
hydrolyzed with 1 M NaOH to give acid 14a quantitatively as
89%) as a pale-yellow oil. 1H NMR (270 MHz, CDCl3)
d 2.31 (s, 3H),
2.70 (dd, J¼4.8, 12.5 Hz, 1H, ABX system), 2.78 (dd, J¼5.4, 12.5 Hz,
1H, ABX system), 3.72 (s, 3H), 4.75 (ddd, J¼4.8, 5.4, 7.9 Hz, 1H,
ABXeXY system), 6.18 (br d, J¼7.9 Hz, 1H, NH, XY system), 6.33 (d,
J¼15.6 Hz, 1H), 7.09 (m, 1H of AA0BB0 system), 7.17e7.41 (complex,
18H), 7.55 (d, J¼15.6 Hz, 1H); 13C NMR (67.8 MHz, CDCl3)
d
21.2 (q),
a yellow solid. 1H NMR (270 MHz, CD3OD)
d
5.91 (d, J¼15 Hz, 1H),
33.9 (t), 51.2 (d), 52.7 (q), 67.0 (s),120.6 (d),121.0 (d),122.9 (d),125.6
(d), 126.9 (d)ꢁ3, 128.0 (d)ꢁ6, 129.5 (d)ꢁ6, 129.8 (d), 136.3 (s), 140.7
(d), 144.3 (s)ꢁ3, 151.0 (s), 164.9 (s), 169.3 (s), 170.9 (s); FT-IR nmax
(cmꢂ1): 3283, 1764, 1739, 1663, 1624; FAB-MS m/z: 566 (MþHþ, 1%),
243 (100). In a similar manner to that used in step h in the synthesis
of analog 2b, the amide obtained above (508 mg, 0.90 mmol) was
cyclized to yield thiazoline ester 11d (93.6 mg, 36%) as a colorless
6.79e6.92 (complex, 4H), 7.38 (d, J¼8.9 Hz, 2H, AA0BB0 system), 7.45
(d, J¼15 Hz, 1H); FT-IR nmax (cmꢂ1): 3311, 1670; EI-MS m/z: 190 (Mþ,
9%), 183 (100). In a similar manner to that used in step g in the
synthesis of analog 2b, acid 14a (32.3 mg, 0.170 mmol) was
condensed with
-cysteine methyl ester derivative, (S)-methyl 2-((2E,4E)-5-
(4-hydroxyphenyl)penta-2,4-dienamido)-3-(tritylthio)propanoate
(44.5 mg, 48%) as a pale-yellow oil. 1H NMR (270 MHz, CDCl3)
2.71
D-Cys(S-Trt)-OMe to afford an N-acyl-S-trityl
D
oil. 1H NMR (270 MHz, CDCl3)
1H, ABX system), 3.65 (dd, J¼9.2, 11.2 Hz, 1H, ABX system), 3.84 (s,
3H), 5.22 (dd, J¼9.2, 9.2 Hz, 1H, ABX system), 7.06e7.10 (complex,
3H), 7.21 (m, 1H), 7.34e7.42 (complex, 2H); 13C NMR (67.8 MHz,
d
2.32 (s, 3H), 3.58 (dd, J¼9.2, 11.2 Hz,
d
(m, 2H, ABX system), 3.71 (s, 3H), 4.72 (m, 1H, ABXeXY system),
5.85 (d, J¼15 Hz, 1H), 6.09 (d, J¼8.0 Hz, 1H, NH, XY system),
6.67e7.46 (complex, 22H); FT-IR nmax (cmꢂ1): 3290, 1739, 1652. In
a similar manner to that used in step h in the synthesis of analog 2b,
the amide obtained above (44.5 mg, 0.08 mmol) was cyclized to
give thiazoline ester 15a (4.6 mg, 20%) as a yellow solid. 1H NMR
CDCl3) d 21.1 (q), 34.7 (t), 52.9 (q), 78.0 (d), 120.6 (d), 122.9 (d), 123.2
(d), 124.9 (d), 129.9 (d), 136.6 (s), 141.1 (d), 151.1 (s), 169.3 (s), 169.8
(s), 171.1 (s); FT-IR nmax (cmꢂ1): 1768, 1743, 1633; EI-MS m/z: 305
(Mþ, 31%), 246 (100). In a similar manner to that used in step i in the
synthesis of analog 2b, thiazoline ester 11d (38.8 mg, 0.13 mmol)
was hydrolyzed with porcine liver esterase to give analog 2d
quantitatively as a yellow powder. Mp 163e165 ꢀC dec; 5% ee from
(270 MHz, CD3OD) d 3.60 (m, 2H, ABX system), 3.79 (s, 3H), 5.21 (dd,
J¼8.9, 8.9 Hz, 1H, ABX system), 6.52 (d, J¼15 Hz, 1H), 6.76 (d,
J¼8.9 Hz, 2H, AA0BB0 system), 6.77e7.18 (m, 3H), 7.38 (d, J¼8.6 Hz,
2H, AA0BB0 system). In a similar manner to that used in step i in the
synthesis of analog 2b, thiazoline ester 15a (4.6 mg, 0.02 mmol)
was hydrolyzed with porcine liver esterase to give analog 3a
quantitatively as a yellow solid. 40% ee from chiral HPLC (retention
chiral HPLC (retention time of
L-isomer: 14.5 min, D-isomer:
16.6 min); 1H NMR (270 MHz, CD3OD)
d
3.54 (dd, J¼8.9, 10.9 Hz, 1H,
ABX system), 3.63 (dd, J¼9.2, 10.9 Hz, 1H, ABX system), 5.05 (dd,
J¼8.9. 9.2 Hz, 1H, ABX system), 6.79 (ddd, J¼1.0, 2.3, 7.9 Hz, 1H), 6.
time of
CD3OD)
L-isomer: 16.2 min, D
-isomer: 15.8 min); 1H NMR (270 MHz,
79e7.23 (complex, 5H); 13C NMR (67.8 MHz, CDCl3)
d
36.5 (t), 81.2
d
3.60 (m, 2H, ABX system), 5.21 (dd, J¼7.3, 7.3 Hz, 1H, ABX
(d), 114.7 (d), 118.1 (d), 120.4 (d), 122.9 (d), 131.0 (d), 137.8 (s), 143.5
(d), 159.1 (s), 171.5 (s), 177.1 (s); FT-IR nmax (cmꢂ1): 3180, 1583, 1628;
EI-MS m/z: 249 (Mþ, 15%), 204 (98), 145 (100). HR-ESI-MS: m/z:
[MþH]þ calcd for C12H12NO3S, 250.0538; found, 250.0493.
system), 6.52 (d, J¼15 Hz, 1H), 6.75e7.56 (complex, 7H), 7.37 (d,
J¼8.9 Hz, 2H, AA0BB0 system); 1FT-IR nmax (cmꢂ1): 3396, 1596; HR-
ESI-MS m/z: [MþH]þ calcd for C14H14NO3S, 276.0694; found,
276.0694, [MþK]þ calcd for C14H13NKO3S, 314.0253; found,
314.0284.
4.2.9. (S)-2-((1E,3E)-4-(4-Hydroxyphenyl)buta-1,3-dienyl)-4,5-
dihydrothiazole-4-carboxylic acid (3a). To a solution of ethyl 4-(tert-
butyldimethylsilyloxy)cinnamate38 (197 mg, 0.64 mmol) in toluene
(2 mL) was added DIBAL (1 M solution in toluene, 3.0 mL, 3 mmol)
and the mixture was stirred at room temperature for 3 h. The re-
action was quenched by adding water (100 mL), and the products
were extracted from the quenched mixture with EtOAc (3ꢁ10 mL).
The organic layers were dried over Na2SO4 and evaporated. The
4.2.10. (S)-4,5-Dihydro-2-[(1E,3E)-4-(4-dimethylaminophenyl)buta-
1,3-dienyl]thiazole-4-carboxylic acid (3b). In a similar manner to
that used in step e in the synthesis of analog 2c, 4-(dimethylamino)
cinnamaldehyde (12b) (504 mg, 2.88 mmol) was subjected to
Wittig olefination to give ester 13b (705 mg, 99%) as a yellow solid.
Mp 115e120 ꢀC; 1H NMR (500 MHz, CDCl3)
d
1.30 (t, J¼7.5 Hz, 3H),
3.00 (s, 6H), 4.20 (q, J¼7.5 Hz, 2H), 5.88 (d, J¼15.5 Hz, 1H),

S. Iwano et al. / Tetrahedron 69 (2013) 3847e3856
3855
6.66e6.71 (m, 3H), 6.82 (d, J¼15.5 Hz, 1H), 7.35 (d, J¼8 Hz, 2H,
AA0BB0 system), 7.44 (dd, J¼12, 15.5 Hz, 1H); 13C NMR (125 MHz,
repeated. The twice-washed precipitates were dissolved in distilled
water containing 0.05% (v/v) trifluoroacetic acid (0.5 mL). The ac-
etone dissolved in the solution was removed under reduced pres-
sure. The resulting aqueous solution was promptly subjected to
HPLC purification just prior to use as described in Section 4.1.2. D-
LH2eAMP was also prepared by essentially the same procedure and
purified by HPLC just prior to use.
CD3OD)
d
14.5 (q), 40.3 (q)ꢁ2, 60.2 (t), 112.1 (d)ꢁ2, 118.3 (d), 121.9
(d), 124.2 (s), 128.7 (d)ꢁ2, 141.2 (d), 145.9 (d), 151.0 (s), 167.7 (s); FT-
IR nmax (cmꢂ1): 1701; ESI-MS m/z: 246 [(MþH)þ]. In a similar
manner to that used in step f in the synthesis of analog 2c, ester 13b
(411 mg, 1.68 mmol) was hydrolyzed with 1 M NaOH to give acid
14b (353 mg, 96%) as a yellow solid. Mp 220e225 ꢀC (lit.39 248 ꢀC);
1H NMR (500 MHz, CD3OD)
d
2.97 (s, 6H), 5.84 (d, J¼15 Hz, 1H), 6.70
4.3.2. Measurements of bioluminescence intensities. Biolumine-
scence intensities of D-LH2, synthesized analogs, and the adenylated
derivatives were measured using an ATTO AB-2200 or AB-2270
luminometer (Hamamatsu, R4220 photomultiplier tube). A re-
(d, J¼9.2 Hz, 2H, AA0BB0 system), 6.73e6.87 (m, 2H), 7.36 (d,
J¼9.2 Hz, 2H, AA0BB0 system), 7.40 (dd, J¼11, 15 Hz, 1H); 13C NMR
(67.8 MHz, CD3OD)
d
39.0 (q)ꢁ2, 111.9 (d)ꢁ2, 118.7 (d), 121.7 (s),
123.1 (d), 128. 6 (d), 130.4 (d), 137.1 (d), 140.6 (s), 145.0 (s), 167.8 (s);
FT-IR nmax (cmꢂ1): 2896, 1684; ESI-MS m/z: 218 [(MþH)þ]. In
a similar manner to that used in step g in the synthesis of analog 2b,
action mixture was prepared by mixing 20
20 L of luciferase solution (0.01 mg/mL), and 20
phosphate buffer (500 mM, pH 8.0). Luminescence reactions were
initiated by injecting 40 L of ATP-Mg (200 M) into the reaction
mixture at ambient temperature. To evaluate bioluminescence ac-
tivity of the adenylated derivatives, 20 L of the adenylated sub-
strate (10 M), 20 L of potassium phosphate buffer (500 mM, pH
8.0), and water (40 L) were mixed, and the luminescence reaction
was initiated by adding 20 L luciferase solution (1 g/mL). In the
mL of substrate (100
mM),
m
m
L of potassium
acid 14b (637 mg, 2.93 mmol) was coupled with
to give an N-acyl-S-trityl -cysteine methyl ester derivative, (S)-
methyl 2-((2E,4E)-5-(4-(dimethylamino)phenyl)penta-2,4-dien-
amido)-3-(tritylthio)propanoate (1.2 g, 69%) as a yellow oil. 1H NMR
(500 MHz, CDCl3) 2.73 (m, 2H, ABX system), 2.99 (s, 6H), 3.71 (s,
D-Cys(S-Trt)-OMe
m
m
D
m
m
m
d
m
3H), 4.77 (ddd, J¼6.8, 6.8, 7.5 Hz, 1H, ABXeXY system), 5.86 (d,
J¼15 Hz, 1H), 6.18 (d, J¼7.5 Hz, 1H, NH), 6.67 (d, J¼8.0 Hz, 2H, AA0BB0
system), 6.70 (d, J¼11.5 Hz, 1H), 6.81 (d, J¼15.5 Hz, 1H), 7.22e7.42
m
m
both cases, light emission was monitored for 180 s with sampling
intervals of 1 s. Emission intensities were expressed as the light
count per second (cps).
(m, 18H); 13C NMR (67.8 MHz, CD3OD)
d
34.3 (t), 39.0 (q)ꢁ2, 53.0
(q), 53.2 (d), 68.3 (s), 98.7 (d)ꢁ2, 112.5 (d), 115.0 (d), 121.1 (s), 124.1
(d), 128.0 (d)ꢁ3, 129.0 (d)ꢁ8, 130.7 (d)ꢁ8, 145.9 (s), 148.0 (s), 158.0
(s), 159.6 (s), 161.0 (s), 172.0 (s); FT-IR nmax (cmꢂ1): 1739, 1593; ESI-
MS m/z: 599 [(MþNa)þ]. In a similar manner to that used in step h
in the synthesis of analog 2b, the amide obtained above (43.3 mg,
0.08 mmol) was cyclized to give thiazoline 15b (17.6 mg, 74%) as
a pale-yellow solid. Mp 145 ꢀC dec; 1H NMR (500 MHz, CDCl3)
4.3.3. Measurements of bioluminescence spectra. Bioluminescence
spectra of synthesized analogs and the adenylated derivatives were
recorded using an ATTO AB-1850 spectrophotometer. A reaction
mixture was prepared by mixing 5
of luciferase solution (1 mg/mL), and 5
buffer (500 mM, pH 8.0). Luminescence reactions were initiated by
injecting 10 L of ATPeMg (200 M) into the reaction mixture.
mL of a substrate (100
mM), 5 mL
mL of potassium phosphate
d
2.99 (s, 6H), 3.55 (m, 2H, ABX system), 3.82 (s, 3H), 5.15 (dd, J¼9.2,
m
m
9.2 Hz, 1H, ABX system), 6.53 (d, J¼15 Hz, 1H), 6.66 (d, J¼9.0 Hz, 2H,
Emission spectra were measured in 1 nm increments from 400 nm
to 750 nm. Bioluminescence emission wavelengths of the adeny-
lated substrates were measured in a similar manner. Luminescence
AA0BB0 system), 6.72 (m, 2H), 6.93 (dd, J¼10, 15 Hz, 1H), 7.34 (d,
J¼9.0 Hz, 2H, AA0BB0 system); 13C NMR (125 MHz, CDCl3)
d 33.7 (t),
39.1 (q)ꢁ2, 51.7 (q), 76.8 (d), 112.0 (d)ꢁ2, 121.0 (d), 122.0 (d), 128.4
(d)ꢁ2, 140.5 (d), 144.8 (d), 151.3 (s), 171.3 (s), 171.3 (s); FT-IR nmax
(cmꢂ1): 1699; ESI-MS m/z: 317 [(MþH)þ]. In a similar manner to
that used in step i in the synthesis of analog 2b, thiazoline ester 15b
(39.9 mg, 0.13 mmol) was hydrolyzed with porcine liver esterase to
give analog 3b (37.9 mg, 99%) as a red solid. Mp 144 ꢀC dec; 70% ee
reactions were initiated by adding 5
mL) to solutions of adenylated substrates (10
potassium phosphate buffer (500 mM, pH 8.0) and 10
m
L of luciferase solution (1 mg/
M, 5 L) in 5 L of
L water.
m
m
m
m
Acknowledgements
from chiral HPLC (OZ-RH column, retention time of
L
-isomer:
The authors are deeply grateful to Dr. Shigeru Nishiyama (Keio
University, Yokohama, Japan) for enthusiastic support and critical
review. This work was supported by Grants-in-Aid for Exploratory
Research (No. 24650633) to S.M. from the Ministry of Education,
Culture, Sports, Science and Technology, and Adaptable & Seamless
Technology Transfer Program through Target-driven R&D (A-Step)
(No. AS2321366E) to S.M. from the Japan Science and Technology
Agency.
9.9 min,
D
-isomer: 12.0 min); 1H NMR (500 MHz, CD3OD)
d 2.97 (s,
6H), 3.58 (m, 2H, ABX system), 5.00 (t, J¼8.9 Hz, 1H, ABX system),
6.49 (d, J¼15 Hz, 1H), 6.70 (d, J¼9.2 Hz, 2H, AA0BB0 system), 6.81 (m,
2H), 7.04 (dd, J¼9.5, 15 Hz, 1H), 7.37 (d, J¼9.2 Hz, 2H, AA0BB0 sys-
tem); 13C NMR (125 MHz, CD3OD)
d
34.5 (t), 39.3 (q)ꢁ2, 78.0 (d),
112.0 (d)ꢁ2, 119.8 (d), 121.9 (d), 124.3 (s), 128.8 (d)ꢁ2, 141.9 (d),
146.2 (d), 151.5 (s), 166.0 (s),172.0 (s); FT-IR nmax (cmꢂ1): 3386,1734,
989; ESI-MS m/z: 303 [(MþH)þ]. HR-ESI-MS m/z: [MþH]þ calcd for
C16H19N2O2S, 303.1167; found, 303.1145.
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