Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Article abstract of DOI:10.1002/chem.201600278

Five new firefly luciferin (1) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains (2–4) and heterocyclic rings derived from amino acids (5 and 6) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin (6), possessing a pyrroline-substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP-carboluciferin (AMP=adenosine monophosphate) and AMP-firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.

Full text of DOI:10.1002/chem.201600278

DOI: 10.1002/chem.201600278
Full Paper
&
Structure–Activity Relationships
Synthesis of Firefly Luciferin Analogues and Evaluation of the
Luminescent Properties
[
a, b]
[a, c]
[d, e]
[f]
[d]
Shuji Ioka,
Tsuyoshi Saitoh,
Satoshi Iwano,
Koji Suzuki, Shojiro A. Maki,
[e]
[b]
[a]
Atsushi Miyawaki, Masaya Imoto, and Shigeru Nishiyama*
Abstract: Five new firefly luciferin (1) analogues were syn-
thesized and their light emission properties were examined.
Modifications of the thiazoline moiety in 1 were employed
to produce analogues containing acyclic amino acid side
chains (2–4) and heterocyclic rings derived from amino acids
luminescence (BL) activity (lmax =547 nm). Results of biolu-
minescence studies with AMP-carboluciferin (AMP=adeno-
sine monophosphate) and AMP-firefly luciferin showed that
the nature of the thiazoline mimicking moiety affected the
adenylation step of the luciferin–luciferase reaction required
for production of potent BL. In addition, BL of 6 in living
mice differed from that of 1 in that its luminescence decay
rate was slower.
(
5 and 6) linked to the benzothiazole moiety. Although
methyl esters of all of the synthetic derivatives exhibited
chemiluminescence activity, only carboluciferin (6), possess-
ing a pyrroline-substituted benzothiazole structure, had bio-
[
2]
2+
Introduction
transformation of firefly luciferin (1) in the presence of Mg
,
adenosine triphosphate (ATP), and O that generates the excit-
2
Bioluminescence (BL)-mediated imaging is a powerful tech-
ed state of oxyluciferin. Excited oxyluciferin then emits yellow–
[
1]
[3]
nique used in both biological and related investigations.
green light (l =553–559 nm) with a high quantum efficien-
max
[
4]
[5]
Among the various processes used for this purpose, the lucifer-
in–luciferase reaction (LLR) has advantageous features related
to its emission properties because it does not require excita-
tion by irradiation with an external light source. BL production
in the LLR system is a consequence of luciferase-catalyzed
cy (f =41%; Scheme 1). Because of its high efficiency and
BL
the availability of firefly luciferase and luciferin, the LLR system
[
6]
has been widely used in biological studies.
[
a] S. Ioka, Dr. T. Saitoh, Prof. Dr. S. Nishiyama
Department of Chemistry, Faculty of Science and Technology
Keio University, Hiyoshi 3-14-1, Kohoku-ku
2
23-8522 Yokohama (Japan)
E-mail: nisiyama@chem.keio.ac.jp
Scheme 1. Four-step BL pathway for LLR-promoted BL involving enzyme
binding, adenylation, oxidative decarboxylation, and light emission.
[
b] S. Ioka, Prof. Dr. M. Imoto
Department of Biosciences and Informatics
Faculty of Science and Technology, Keio University
Hiyoshi 3-14-1, Kohoku-ku, 223-8522 Yokohama (Japan)
The mechanistic pathway for the reaction catalyzed by firefly
luciferase, leading to BL, has been fully elucidated. In addition,
many investigations have been carried out to expand the sub-
[
c] Dr. T. Saitoh
International Institute for Integrative Sleep Medicine (WPI-IIS)
University of Tsukuba, Tennodai 1-1-1, Tsukuba-si
[
7]
3
05-8577 Ibaraki (Japan)
strate scope of this process. The results of these studies
showed that the generation of BL required the use of sub-
strates that, similar to 1, had an a-amino acid center with the
[
d] Dr. S. Iwano, Dr. S. A. Maki
Department of Engineering Science
The University of Electro-Communications
Chofugadake 1-5-1, Chofu, 182-8585 Tokyo (Japan)
[8]
d-absolute configuration. In particular, aminoluciferin, in
which the phenolic hydroxyl group of the parent substrate
[
e] Dr. S. Iwano, Prof. Dr. A. Miyawaki
Laboratory for Cell Function Dynamics
Brain Science Institute, RIKEN
[
9]
was replaced by an NH group, was found to have BL activity.
2
In an earlier study, we demonstrated that the emission wave-
length maxima for BL by analogues of 1 were dependent on
the nature of the benzothiazole ring. For instance, incorpora-
tion of a more extensively conjugated chain in this moiety in-
Hirosawa 2-1, Wako, 351-0198 Saitama (Japan)
[
f] Prof. Dr. K. Suzuki
Department of Applied Chemistry
Faculty of Science and Technology, Keio University
Hiyoshi 3-14-1, Kohoku-ku, 223-8522 Yokohama (Japan)
[
10]
duces a redshift in the luminescence wavelength.
Importantly, analogues of 1 that display redshifted BL are
suitable for use in noninvasive, whole-body imaging systems
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201600278.
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because they generate near-IR light that is more capable of
penetrating thick tissues than short-wavelength light arising
from the LLR. The investigation described below was guided
by the goal of expanding the substrate range of the luciferase-
promoted reaction and to produce new substances that effi-
ciently produced BL. In these effort, substances were prepared
and explored in which groups of the thiazoline moiety of
of 10 with acid hydrochlorides of glycine, d-alanine, and d-
serine, followed by removal of the MOM group in each amide
by using TFA, formed the respective ester derivatives 11–13. Fi-
nally, treatment of 11–13 with a solution of NaOH in methanol
[
14]
generated the respective benzothiazoylamino acids 2–4.
Preparation of the oxazoline benzothiazole 5 began with se-
lective acetylation of the phenolic hydroxyl group of the
serine-derived intermediate 13 (Scheme 3). This process pro-
1
were replaced with those derived biosynthetically from d-
[
5]
cysteine. The results of earlier studies of luciferin analogues
in which the sulfur atom of the thiazoline ring was replaced by
an selenium atom, and NH and Me C groups, have been re-
2
[
11]
ported.
Herein, several novel analogues are described in
which the thiazoline ring in 1 is replaced by glycine, d-alanine,
d-serine side chains (2–4; Figure 1), d-oxazoline, and d-pyrro-
Scheme 3. Synthesis of oxazoline benzothiazole 5. DAST=diethylaminosul-
fur trifluoride.
duced monoacetate 14 in only low yield because of competi-
tive esterification of the primary alcohol group in 13. Impor-
tantly, the direct cyclization reaction of unprotected phenol 13
did not take place cleanly. Treatment of 14 with DAST promot-
ed the desired cyclization to generate oxazoline 15, which un-
derwent sequential lipase-promoted deacetylation and methyl
ester hydrolysis to form the oxazoline ring-containing luciferin
analogue 5.
Figure 1. Firefly luciferin (1) is biosynthetically produced by the condensa-
tion of d-cycteine and 2-cyano-6-hydroxybenzothiazole. Luciferin analogues
2
–6 derived from amino acids prepared in this study.
line rings (5 and 6; Figure 1). All compounds were evaluated
to explain the role of the thiazoline moiety in BL. The results of
BL studies showed that the nature of the thiazoline mimicking
moiety affected the adenylation step of the LLR required for
the production of potent BL. Moreover, BL of one of the newly
prepared derivatives in living mice was shown to differ from
that of 1 in that its decay rate was lower.
In the pathway for the synthesis of carboluciferin (6;
[
15]
Scheme 4), bromide 17 was generated through Sandmeyer
reaction of a commercially available aminothiazolidine 16. Re-
moval of the methoxy group in 17 gave the corresponding
phenol 18, which was protected by using TBSCl to give the
phenylsilyl ether 19. To generate the pyrrolidinone 22, which
was used in a coupling reaction with 19, the pyrrolidione car-
[
16]
boxylate 20 was transformed into tert-butyl ester 21. Protec-
Results and Discussion
[16]
tion of 21 with Boc gave 22, which reacted with the lithium
organometallic derivative of 19, produced by treatment with
nBuLi, to generate the glutamate-linked benzothioazole 23.
The route used for the synthesis of the benzothiazoylamino
acids 2–4 was initiated by the pyridine hydrochloride promot-
ed thermal transformation of 2-cyano-6-methoxybenzothiazole
[12]
(
7) to form phenol 8 (Scheme 2). Conversion of the nitrile
[
13]
group in 8 into the corresponding methyl ester in 9,
by
treatment with MeOH/K CO , was followed by MOM protection
2
3
of the phenolic hydroxyl group to give 10. Coupling reactions
Scheme 4. Synthesis of carboluciferin (6). isoAmONO=isoamyl nitrite,
TBSCl=tert-butyldimethylsilyl chloride, Boc=tert-butyloxycarbonyl,
DMAP=4-dimethylaminopyridine, TBAF=tetrabutylammonium fluoride.
Scheme 2. Synthesis of benzothiazoylamino acids 2–4. MOM=methoxy-
methyl, TFA=trifluoroacetic acid.
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TBAF-mediated desilylation of 23 formed the free phenol 24,
which then underwent TFA-promoted cyclization to produce
linked pyrroline benzothiazole 6.
stances as inhibitors of the luciferase-catalyzed reaction of
1 were determined by means of Lineweaver–Burk analysis
[
19]
(Table S1 and Figure S3 in the Supporting Information). l-
Firefly luciferin (l-1) was utilized as a positive control because
[7]
Luciferase-promoted BL was evaluated by using 50 mm so-
lutions of the luciferin analogues 2–6 in potassium phosphate
buffer (pH 6.0). The results showed that only 6 produced a BL
response, whereas benzothiazoylamino acids 2–4 and oxazo-
line 5 analogues did not (Figure 2 and Table S1 in the Support-
ing Information). The total photon yield of the luciferase-cata-
[
5c]
it was known to inhibit BL similar to that of 1. The results
demonstrated that alanine derivative 3 and oxazoline analogue
5 bound to luciferase, whereas related substances 2 and 4 did
not. On the basis of these observations, the cyclic structure of
the thiazoline moiety was important for the recognition of fire-
fly luciferase (alanine derivative 3 could not be explained). We
propose that the thiazoline moiety in 1 is a key structural re-
quirement for substances that participate in the LLR leading to
BL (Figure 2).
5
lyzed reaction of 6 (1.52”10 ) was 0.56% of the process with
7
1
as a substrate (2.72”10 ). In addition, the maximum emis-
sion wavelength of 6 BL was blueshifted (lmax =547 nm) rela-
tive to that of 1. To assess the binding affinity of synthetic luci-
ferin 6 to luciferase, the Michaelis constant (K , defined as the
concentration at half of the maximum reaction rate) was deter-
The BL activity of AMP-carboluciferin (AMP=adenosine
monophosphate) was explored next to clarify why the BL activ-
ity of 6 was lower than that of 1. AMP-carboluciferin and AMP-
firefly luciferin were prepared by using a procedure reported
m
[
10,17]
mined.
The Km value for 6 ((82.5Æ7.2) mm) was smaller
than that of 1 ((116Æ22.0) mm; Figure S1 in the Supporting In-
formation), which showed that the binding affinity of 6 to luci-
ferase was higher than that of 1. The Km value of 1 was differ-
ent from that previously reported because of the low ATP-
[
7,11c]
in the literature,
and purified by means of HPLC (Figure S4
in the Supporting Information). The luciferase solution used
was diluted 10-fold because the photon emission of AMP-fire-
fly luciferin exceeded the limit of the luminometer. The BL ac-
2
+
Mg concentrations (see the Experimental Section).
7
Next, the chemiluminescence properties of methyl esters of
tivity of AMP-carboluciferin (4.75”10 ) was 13000 times great-
[18]
3
the synthetic derivatives were examined. For this purpose,
tBuOK (80 mL) in DMSO (100 mm) was added to solutions of
the individual luciferin analogues in DMSO (1 mm, 20 mL) at
room temperature. Light emission was monitored for 60 s at
sampling intervals of 0.1 s. The results showed that the methyl
esters of 2–6 generated chemiluminescence with nearly identi-
cal efficiencies (Figure S2 in the Supporting Information).
er than that of 6 (3.54”10 ). In contrast, the bioluminescent
8
activity of AMP-firefly luciferin (2.11”10 ) was only 285 times
5
greater than that of 1 (7.42”10 ; Figure 3). These observations
suggested that the low BL activity of 6 was a consequence of
the low efficiency of the adenylation step in the LLR pathway.
In the final phase of this investigation, the ability of 6 to dis-
play BL in CAG-ffLuc-cp156 transgenic mice, developed in our
[
20]
The results appear to show that the structures of the groups
mimicking the thiazoline ring in 1 influence the BL yield as
a consequence of their effect on binding to and/or reacting
with the enzyme. Information was gained to show why 2–5
did not display BL activities, despite having chemilumines-
previous research, was explored.
Compounds 1 and 6
(5 mm; 100 mL in sterile water) were independently injected
into the abdominal cavities of mice (n=3). The results of in
vivo imaging showing the change in BL with time are dis-
played in Figure 4. Carboluciferin (6) displayed BL in a mouse
that increased with time and peaked at 2400 s following injec-
cence activities. For this purpose, the K values of these sub-
i
Figure 2. Change in the BL properties of a) 6 (blue) and b) 1 (green) with time; c) BL spectra. Compounds 3–5 did not show BL.
Chem. Eur. J. 2016, 22, 9330 – 9337 www.chemeurj.org ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 3. Change in BL activities of AMP-carboluciferin (a) and AMP-firefly luciferin (b); c) BL spectra.
lived BL was displayed by 6 than that of 1 following injection
into live mice. The observations made in this investigation pro-
vide information that could contribute to the design of sub-
strates that undergo improved luciferase-promoted BL.
Experimental Section
General
IR spectra were recorded on a JASCO Model FT/IR-4200 spectro-
1
13
photometer. H and C NMR spectra were obtained on JEOL JNM
AL-400, JEOL JNM ECX-400, and JEOL JNM a-400 spectrometers as
Figure 4. In vivo imaging showing changes in BL as a function of time fol-
lowing injection of mice with 6 (blue) and 1 (green).
solutions in CDCl with tetramethylsilane as the internal standard.
3
HRMS results were obtained on a Waters LCT Premier XE (ESI) spec-
trometer. Preparative and analytical TLC were carried out on silica-
gel plates (Kieselgel 60 F254, E. Merck AG, Germany), and UV (l=
tion. In comparison, BL from 1 increased more rapidly with
time and reached a maximum at 300 s after injection.
2
54 nm) light and 5% phosphomolybdic acid in ethanol were used
as developers. Kanto Chemical silica gel 60N (spherical, neutral,
3–210 mm) was used for column chromatography. All reactions
6
Conclusion
were carried out under an argon atmosphere. When necessary, sol-
vents were dried prior to use. Anhydrous solvents were purchased
from Kanto Chemical Co. Inc. and stored over 4 Š molecular sieves
under an argon atmosphere.
We synthesized new luciferin analogues containing acyclic d-
amino acid side chains (2–4) and their cyclic derivatives de-
rived from d-amino acid (5 and 6) linked to the benzothiazole
ring system of the parent compound. Evaluation of these sub-
stances showed that 6 had only 0.5% of the luciferase-promot-
ed BL activity of 1, and that the benzothiazoylamino acids and
oxazoline luciferin analogues did not display BL in the pres-
ence of this enzyme. Observations made in an additional study
showed that ester derivatives of these substances were chemi-
luminescent. An evaluation of BL of AMP-carboluciferin and
AMP-firefly luciferin revealed that the activity of the former
substance was 13000 times larger than that of 6, whereas the
activity of AMP-firefly luciferin was only 285 times larger than
that of 1. These results indicated that the thiazoline-mimicking
moiety in 6 affected the adenylation step of LLR, whereas the
benzothiazole moiety played an important role in determining
the luminescence intensity and wavelength. Finally, longer
Determination of luminescent activities
À1
A 1 mgmL stock solution of commercially available luciferase
(
Promega (QuantiLum recombinant Photinus pyralis luciferase,
E1701) and Sigma (L9506)) in 50 mm Tris-HCl buffer (pH 8.0) con-
taining 10% glycerol was prepared and stored at À808C. Prior to
use, the luciferase stock solution was diluted 100-fold with 50 mm
potassium phosphate buffer (pH 6.0) containing 35% glycerol. The
diluted luciferase solution was cooled on ice until required. ATP-
2
+
Mg was purchased from Nacalai Tesque, and buffer chemicals
were obtained from Wako Chemicals or Kanto Chemicals. A stock
solution of the synthesized analogue (5 mm) in 50 mm potassium
phosphate buffer (pH 6.0) was prepared and stored at À808C. De-
ionized water (Millipore, Milli-RX-12a) was used to prepare aqueous
assay solutions. The pH values of the solutions were determined
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with a Horiba F-23 pH meter. Chemiluminescence and BL intensi-
ties were measured by using ATTO AB-2200 or AB-2700 luminome-
ters (Hamamatsu, R4220 photomultiplier tube). Chemiluminescence
and BL wavelength spectra were recorded on an ATTO AB-1850
spectrophotometer.
In vivo imaging
For BL measurements, 37-week-old CAG-ffLuc-cp156 transgenic
mice were grown. A mouse was injected with a mixture of somno-
À1
pentyl (30 mgkg ) and luciferin (5 mm, 100 mL in sterile water)
into its abdominal cavity. Immediately after injection, the scans
were performed with an in vivo imaging system (LumazoneFA,
Roper Japan). Light emission was monitored for 2700 s at exposure
times of 0.5 (1) and 60 s (6). The experimental procedures and
housing conditions for animals were approved by the Institutes
Animal Experiments Committee at RIKEN and all animals were
cared for and treated in accordance with the Institutional Guide-
lines for Experiments Using Animals. Synthesis of new compounds
NMR spectra of new compounds are shown in the Supporting In-
formation.
Chemiluminescence activity
Methyl ester derivatives were synthesized to evaluate chemilumi-
nescence. Chemiluminescence measurements were made by
adding a solution of tBuOK (80 mL) in DMSO (100 mm) into solu-
tions of the methyl esters of the luciferin analogues in DMSO
(
1 mm, 20 mL) at room temperature. Light emission was monitored
for 60 s at sampling intervals of 0.1 s. Emission intensities were ex-
pressed as the light count per 0.1 s. Spectra were recorded from
l=400 to 750 nm in 1 nm increments.
Methyl 6-(methoxymethoxy)benzo[d]thiazole-2-carboxylate
(10)
BL activity
NaH (60 mg, 1.50 mmol, 60% dispersion in mineral oil) and MOMCl
(0.51 mL, 6.75 mmol) were added to a stirred solution of 9
(284 mg, 1.35 mmol) in DMF (5 mL). The resulting mixture was
Solutions for BL measurements were prepared by mixing the luci-
ferin derivatives (20 mL; 100 mm), firefly luciferase solution (20 mL;
À1
0
.01 mgmL ), and potassium phosphate buffer (20 mL; 500 mm,
stirred at room temperature for 30 min, diluted with H O (10 mL),
2
2
+
pH 8). The enzymatic reactions were initiated by adding ATP-Mg
and extracted with CHCl . The organic layers were combined,
3
(
40 mL; 200 mm) to these solutions at room temperature. A low
washed with brine, dried over anhydrous Na SO , and concentrated
in vacuo. The residue was subjected to column chromatography
2
4
2
+
ATP-Mg
concentration was used because high concentrations
gave a strong background signal. Light emission was monitored
for 30 s at sampling intervals of 0.1 s. Measurements of emission
intensities and wavelengths were made by using the same tech-
niques as those employed for chemiluminescence measurements.
on silica gel (EtOAc/hexane, 1/2) to give 10 (207.5 mg, 61%) as
1
a yellow oil. H NMR (CDCl ): d=8.08 (d, J=9.2 Hz, 1H; Ar), 7.57 (d,
3
J=2.5 Hz, 1H; Ar), 7.23 (dd, J=2.5, 9.2 Hz, 1H; Ar), 5.23 (s, 2H;
OCH OCH ), 4.03 (s, 3H; COOCH ), 3.48 ppm (s, 3H; OCH OCH );
2
3
3
2
3
13
C NMR (CDCl ): d=161.2 (COOCH ), 157.2 (Ar), 155.9 (Ar), 148.5
3
3
The Km values of the luciferase-catalyzed reactions of 1 and 6 were
determined by using the initial maximum light intensities to esti-
mate initial velocities. Reactions were initiated by injection of
(Ar), 138.5 (Ar), 126.2 (Ar), 118.7 (Ar), 106.9 (Ar), 94.8 (OCH
OCH ),
2 3
À1
56.3 (OCH OCH ), 53.6 ppm (COOCH ); IR (film): n˜ =1743 cm ;
2
3
3
+
HRMS (ESI): m/z calcd for C11H12NO S [M +H]: 254.0487; found:
4
2
+
2
00 mm ATP-Mg into a mixture of 100 mm potassium phosphate
254.0486.
À1
buffer containing 1–500 mm 1 and 6 and 20 mgmL firefly lucifer-
ase (P. pylalis, Promega Corp). For the K determinations, reactions
i
Methyl 2-(6-hydroxybenzo[d]thiazole-2-carboxamido)acetate
11)
2
+
were initiated by the injection of 200 mm ATP-Mg into a mixture
of 100 mm potassium phosphate buffer containing 1–500 mm of lu-
(
À1
ciferin, 1–500 mm of analogues, and 20 mgmL firefly luciferase.
Glycine methyl ester hydrochloride (286 mg, 2.28 mmol) and Et N
3
(
0.63 mL, 4.56 mmol) were added to a stirred solution of 10
[7,11c]
Following a procedure reported in the literature,
we prepared
(57.7 mg, 0.228 mmol) in MeOH (4 mL). The mixture was stirred at
room temperature for 19 h, diluted with a saturated aqueous solu-
and purified AMP-firefly luciferin and AMP-carboluciferin immedi-
ately prior to their use. For this purpose, a solution of dicyclohexyl-
carbodiimide (DCC; 20 mg, 0.097 mmol) in DMSO (0.8 mL) was
added to a solution of 1 or 6 (1 mg, 3.81 mmol) and (À)-adenosine-
tion of NH
Cl (20 mL), and extracted with CHCl
. The organic layers
SO
4
3
were combined, washed with brine, dried over anhydrous Na
,
4
2
and concentrated in vacuo. The residue was dissolved in TFA
(2 mL). After being stirred for 30 min., the mixture was concentrat-
ed in vacuo. The residue was subjected to column chromatography
5
0
’-monophosphoric acid (AMP; free acid, Oriental Yeast Co.; 10 mg,
.029 mmol) in DMSO (0.5 mL) under an argon atmosphere. The
mixture was stirred vigorously for 10 min at room temperature and
then acetone (1.5 mL) was added to quench the reaction. The
white precipitate formed was separated by centrifugation and the
supernatant was discarded. The precipitates were suspended in
ice-cold acetone (1 mL) and then centrifuged. The washing opera-
tion was repeated. The twice-washed precipitates were dissolved
in distilled water containing 0.05% (v/v) TFA (0.5 mL). Acetone, dis-
solved in the solution, was removed under reduced pressure and
the resulting aqueous solution was promptly subjected to HPLC
purification immediately prior to use (Figure S4 in the Supporting
Information). To evaluate their bioluminescent activities, luciferase
on silica gel (EtOAc/hexane, 1/1) to give 11 (28.1 mg, 46%) as
1
a yellow powder. M.p. 215–2178C; H NMR ([D
]acetone): d=8.48
6
(br, 1H; NH), 7.92 (d, J=9.0 Hz, 1H; Ar), 7.53 (d, J=2.5 Hz, 1H; Ar),
7.16 (dd, J=2.5, 9.0 Hz, 1H; Ar), 4.23 (d, J=6.3 Hz, 2H; CH ),
]acetone): d=170.5
), 158.2 (Ar), 157.4 (Ar), 147.7 (Ar), 139.5
2
13
3.73 ppm (s, 3H; COOCH
(CONH), 161.1 (COOCH
); C NMR ([D
6
3
3
(Ar), 126.0 (Ar), 118.2 (Ar), 107.7 (Ar), 52.4 (COOCH
), 41.8 ppm
3
À1
(CH
); IR (KBr): n˜ =3265, 1734, 1654 cm ; HRMS (ESI): m/z calcd for
2
+
C
H N O S [M +H]: 267.0440; found: 267.0437.
11 2 4
11
2-(6-Hydroxybenzo[d]thiazole-2-carboxamido)acetic acid (2)
À1
solution (20 mL; 1 mgmL ) was added to a solution of the adeny-
lated substrates (20 mL; 100 mm), potassium phosphate buffer
A 25 mm aqueous solution of NaOH (5 mL) was added to a stirred
solution of 11 (12.0 mg, 0.0432 mmol) in MeOH (1 mL). The result-
ing mixture was stirred at room temperature for 25 min, diluted
with 2m HCl (0.1 mL), and extracted with EtOAc. The organic layers
(
20 mL; 500 mm, pH 8.0), and water (40 mL). Light emission was
monitored for 180 s at sampling intervals of 1 s. Emission intensi-
ties were expressed in light counts per second (cps).
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were combined, washed with brine, dried over anhydrous Na SO ,
(Ar), 148.1 (Ar), 140.0 (Ar), 126.3 (Ar), 118.4 (Ar), 107.5 (Ar), 62.8
2
4
and concentrated in vacuo to give 2 (11.3 mg, quant) as a white
(CHCH OH), 56.4 ppm (CHCH OH); IR(KBr): n˜ =3388, 2942, 1729,
1652 cm ; HRMS (ESI): m/z calcd for C H N O S [M +H]:
11 11 2 5
2
2
1
À1
+
powder. M.p. 210–2128C; H NMR (CD OD): d=7.93 (d, J=9.3 Hz,
3
1
4
H; Ar), 7.36 (d, J=2.4 Hz, 1H; Ar), 7.07 (dd, J=2.4, 9.3 Hz, 1H; Ar),
283.0389; found: 283.0379.
1
3
.15 ppm (s, 2H; CH2); C NMR (CD OD): d=172.5 (CONH), 162.6
3
(
1
1
2
COOH), 160.4 (Ar), 158.7 (Ar), 148.2 (Ar), 140.0 (Ar), 126.3 (Ar),
Methyl (R)-2-(6-acetoxybenzo[d]thiazole-2-carboxamido)-3-
hydroxypropanoate (14)
18.3 (Ar), 107.4 (Ar), 42.0 ppm (CH ); IR (KBr): n˜ =3379, 3093, 1736,
2
À1
+
644 cm
; HRMS (ESI): m/z calcd for C H N O S [M +H]:
10 9 2 4
53.0283; found: 253.0291.
Acetic anhydride (0.11 mL, 1.02 mmol) and NaHCO3 (103.0 mg,
1
0
.23 mmol) were added to a stirred solution of 13 (132.1 mg,
.407 mmol) in THF (10 mL). The mixture was stirred at room tem-
(
R)-2-(6-Hydroxybenzo[d]thiazole-2-carboxamido)propanoic
perature for 1 h, diluted with a saturated aqueous solution of
NH Cl (30 mL), and extracted with CHCl . The organic layers were
acid (3)
4
3
combined, washed with brine, dried over anhydrous Na SO , and
concentrated in vacuo. The residue was subjected to column chro-
2
4
Compound 10 (69.6 mg, 0.274 mmol) in MeOH (4 mL) was treated
with d-alanine methyl ester hydrochloride (279 mg, 2.00 mmol)
matography on silica gel (EtOAc/hexane, 1/1) to give 14 (109.4 mg,
and Et N (0.55 mL, 4.00 mmol) in the same procedure as that de-
3
25
D
7
9%) as a white powder. M.p. 138–1418C; [a] =À16.6 (c=1.00,
scribed for 11 to give 12 (60.2 mg, 84%) as a white powder. M.p.
1
2
D
5
1
CHCl
3
); H NMR (CDCl
3
): d=8.26 (d, J=7.8 Hz, 1H; NH), 8.02 (d, J=
1
7
2
72–1758C; [a] =À15.6 (c=1.00, CH OH); H NMR (CD OD): d=
3
3
8
1
.8 Hz, 1H; Ar), 7.68 (d, J=2.0 Hz, 1H; Ar), 7.26 (dd, J=2.0, 8.8 Hz,
H; Ar), 4.87 (ddd, J=3.9, 7.8 Hz, 1H; CHCH OH), 4.16 (dd, J=3.9,
.90 (d, J=9.0 Hz, 1H; Ar), 7.33 (d, J=2.5 Hz, 1H; Ar), 7.06 (dd, J=
.5, 9.0 Hz, 1H; Ar), 4.65 (q, J=7.2 Hz, 1H; CHCH ), 3.77 (s, 3H;
2
3
1
3
11.7 Hz, 1H; CHCH
OH), 4.08 (dd, J=3.9, 11.7 Hz, 1H; CHCH OH),
2 2
COOCH ), 1.53 ppm (d, J=7.2 Hz, 3H; CHCH ); C NMR (CD OD):
3
3
3
13
3
.82 (s, 3H; COOCH ), 2.35 ppm (s, 3H; ArOCOCH ); C NMR
3
3
d=174.1 (CONH), 162.0 (COOCH ), 160.0 (Ar), 159.4 (Ar), 147.9 (Ar),
3
(
CDCl ): d=170.4 (CONH), 169.5 (ArOCOCH ), 163.1 (COOCH ), 160.0
3 3 3
1
40.0 (Ar), 126.2 (Ar), 118.7 (Ar), 107.5 (Ar), 53.0 (COOCH ), 49.8
3
À1
(Ar), 150.1 (Ar), 149.5 (Ar), 137.8 (Ar), 125.2 (Ar), 121.9 (Ar), 115.1
(CHCH ), 17.5 ppm (CHCH ); IR (KBr): n˜ =3286, 1733, 1652 cm
;
3
3
+
(Ar), 63.1 (CHCH
2
OH), 55.1 (CHCH
2
OH), 53.1 (COOCH
3
), 21.3 ppm
HRMS (ESI): m/z calcd for C H N O S [M +H]: 281.0596; found:
1
2
13
2
4
À1
(
ArOCOCH ); IR (KBr): n˜ =3402, 1752, 1743, 1653 cm ; HRMS (ESI):
3
2
81.0601.
+
m/z calcd for C H N O S [M +H]: 339.0626; found: 339.0651.
14
15
2
6
Alkaline hydrolysis of 12 (17.3 mg, 0.0624 mmol), as in the case of
, provided 3 (11.3 mg, quant) as a white powder. M.p. 174–1768C;
Methyl (R)-2-(6-acetoxybenzo[d]thiazol-2-yl)-4,5-dihydrooxa-
zole-4-carboxylate (15)
2
25
1
[
9
1
a] =À24.4 (c=1.00, CH OH); H NMR (CD OD): d=7.93 (d, J=
D
3
3
.3 Hz, 1H; Ar), 7.35 (d, J=2.4 Hz, 1H; Ar), 7.07 (dd, J=2.4, 9.3 Hz,
DAST (0.42 mL, 3.17 mmol) was added to a stirred solution of 14
H; Ar), 4.62 (q, J=7.3 Hz, 1H; CHCH ), 1.56 ppm (d, J=7.3 Hz, 3H;
3
(
109.4 mg, 0.323 mmol) in CH Cl (110 mL). The resulting mixture
2 2
1
3
CHCH3); C NMR (CD OD): d=173.0 (CONH), 161.8 (COOH), 160.6
3
was stirred at À808C for 20 min, diluted sequentially with saturat-
(
Ar), 158.7 (Ar), 148.2 (Ar), 139.9 (Ar), 126.2 (Ar), 118.3 (Ar), 107.4
ed aqueous solutions of NaHCO (30 mL) and NH Cl (50 mL), and
3
4
(
1
2
Ar), 49.8 (CHCH ), 17.8 ppm (CHCH ); IR (KBr): n˜ =3388, 2942, 1729,
3
3
extracted with CHCl . The organic layers were combined, washed
À1
+
3
652 cm
; HRMS (ESI): m/z calcd for C H N O S [M +H]:
11 11 2 4
with brine, dried over anhydrous Na SO , and concentrated in
2
4
67.0440; found: 267.0420.
vacuo. The residue was subjected to column chromatography on
silica gel (EtOAc/hexane, 1/1) to give 15 (95.2 mg, 90%) as a white
2
D
5
1
powder. M.p. 168–1728C; [a] =À14.1 (c=1.00, CHCl ); H NMR
(
R)-3-Hydroxy-2-(6-hydroxybenzo[d]thiazole-2-carboxamido)-
3
(
CDCl ): d=8.16 (d, J=9.0 Hz, 1H; Ar), 7.72 (d, J=2.2 Hz, 1H; Ar),
3
.28 (dd, J=2.2, 9.0 Hz, 1H; Ar), 5.07 (dd, J=8.3, 10.8 Hz, 1H;
propanoic acid (4)
7
Compound 10 (232 mg, 0.916 mmol) in MeOH (10 mL) was treated
with d-serine methyl ester hydrochloride (1.01 g, 6.39 mmol) and
Et N (1.8 mL, 12.8 mmol) in the same procedure as that described
3
CHCH O), 4.87 (dd, J=8.3, 8.8 Hz, 1H; CHCH OH), 4.77 (dd, J=8.8,
2
2
10.8 Hz, 1H; CHCH OH), 3.84 (s, 3H; COOCH ), 2.51 ppm (s, 3H; Ar-
2
3
13
OCOCH3); C NMR (CDCl ): d=170.6 (NCO), 169.4 (ArOCOCH ),
3 3
for 11 to give 13 (161 mg, 64%) as a white powder. M.p. 181–
161.1 (COOCH ), 155.1 (Ar), 151.1 (Ar), 149.8 (Ar), 137.0 (Ar), 125.7
3
2
5
1
1
838C; [a] =À4.7 (c=1.00, CH OH); H NMR (CD OD): d=7.91 (d,
(Ar), 121.9 (Ar), 114.7 (Ar), 71.1 (NCHCH O), 68.9 (NCHCH O), 53.1
D
3
3
2
2
J=9.0 Hz, 1H; Ar), 7.34 (d, J=2.5 Hz, 1H; Ar), 7.07 (dd, J=2.5,
.0 Hz, 1H; Ar), 4.74 (t, J=4.0 Hz, 1H; CHCH OH), 4.06 (dd, J=4.0,
(COOCH3), 21.3 ppm (ArOCOCH3); IR (KBr): n˜ =1740, 1731,
À1
+
9
1635 cm
; HRMS (ESI): m/z calcd for C H N O S [M +H]:
14 13 2 5
2
1
3
1
1.4 Hz, 1H; CHCH OH), 3.97 (dd, J=4.0, 11.4 Hz, 1H; CHCH OH),
321.0545; found: 321.0521.
2
2
1
3
.80 ppm (s, 3H; COOCH3); C NMR (CD OD): d=171.8 (CONH),
3
62.1 (COOCH ), 159.8 (Ar), 159.6 (Ar), 147.8 (Ar), 140.1 (Ar), 126.2
3
(R)-2-(6-Hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrooxazole-4-
(
Ar), 117.6 (Ar), 107.6 (Ar), 62.8, (CHCH OH), 56.4 (CHCH OH),
2 2
À1
carboxylic acid (5)
5
3.1 ppm (COOCH ); IR (KBr): n˜ =3373, 3262, 1749, 1653 cm ;
3
+
HRMS (ESI): m/z calcd for C H N O S [M +H]: 297.0545; found:
A 100 mm aqueous solution of NH HCO3 (10 mL) was added to
1
2
13
2
5
4
2
97.0569.
a stirred solution of 15 (32.0 mg, 0.0992 mmol) and Lipase PS IM
Amano (200 mg) in isopropyl ether (10 mL). The resulting mixture
was stirred at 508C for 6 h and concentrated in vacuo. The residue
was filtered to give 5 (95.2 mg, 91%) as a brown powder. M.p.
Alkaline hydrolysis of 13 (12.8 mg, 0.0462 mmol), as in the case of
, provided 4 (10.1 mg, quant) as a white powder. M.p. 171–1728C;
2
25
1
25
1
[
a] =À8.9 (c=1.00, CH OH); H NMR (CD OD): d=7.94 (d, J=
140–1438C; [a] =À2.89 (c=1.00, CH OH); H NMR (CD OD): d=
D
3
3
D
3
3
9
1
1
.3 Hz, 1H; Ar), 7.36 (d, J=2.4 Hz, 1H; Ar), 7.08 (dd, J=9.3, 2.4 Hz,
H; Ar), 4.69 (t, J=3.9 Hz, 1H; CHCH OH), 4.07 (dd, J=3.9, 11.2 Hz,
H; CHCH OH), 3.99 ppm (dd, J=3.9, 11.2 Hz, 1H; CHCH OH);
C NMR (CD OD): d=172.9 (CONH), 161.9 (COOH), 160.4 (Ar), 158.8
7.89 (d, J=8.8 Hz, 1H; Ar), 7.34 (d, J=2.4 Hz, 1H; Ar), 7.07 (dd, J=
2.4, 8.8 Hz, 1H; Ar), 4.88 (t, J=8.8 Hz, 1H; CHCH O), 4.75–4.68 ppm
2
2
13
(m, 2H; NCHCH O); C NMR (CD OD): d=177.5 (NCO), 161.3
2 3
2
2
1
3
(COOH), 159.0 (Ar), 153.1 (Ar), 147.7 (Ar), 139.0 (Ar), 125.8 (Ar),
3
Chem. Eur. J. 2016, 22, 9330 – 9337
www.chemeurj.org
9335 ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
1
18.5 (Ar), 107.2 (Ar), 73.6 (NCHCH O), 72.1 ppm (NCHCH O); IR
sulting mixture was stirred at À788C for 10 min and diluted by the
addition of 22 (558 mg, 1.96 mmol) in dry THF (10 mL) at À788C.
The resulting mixture was stirred at À408C for 2 h, diluted with
a saturated aqueous solution of NaHCO3 (50 mL), and extracted
2
À1
2
(
KBr): n˜ =3287, 2960, 1729, 1647 cm ; HRMS (ESI): m/z calcd for
+
C H N O S [M +H]: 265.0265; found: 265.0285.
11
9
2
4
with CHCl . The organic layers were combined, washed with brine,
3
Methyl 2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrooxa-
zole-4-carboxylate (25)
dried over anhydrous Na SO , and concentrated in vacuo. The resi-
2
4
due was subjected to column chromatography on silica gel
NaHCO (10.5 mg, 0.125 mmol) was added to a stirred solution of
(EtOAc/hexane, 1/5) and gel permeation chromatography (AIGEL-
3
À1
1
5 (13.4 mg, 0.0416 mmol) in MeOH (5 mL). The resulting mixture
1H, CHCl
3
, flow rate 3.5 mLmin , UV detection at l=254 nm) to
25
was stirred at room temperature for 2 h, diluted with a saturated
give 23 (491 mg, 52%) as a yellow oil. [a] =À7.16 (c=1.00,
D
1
aqueous solution of NH Cl (30 mL), and extracted with EtOAc. The
CHCl
3
); H NMR (CDCl
3
): d=7.99 (d, J=9.0 Hz, 1H; Ar), 7.34 (d, J=
4
organic layers were combined, washed with brine, dried over anhy-
drous Na SO , and concentrated in vacuo. The residue was subject-
ed to column chromatography on silica gel (EtOAc/hexane, 1/1) to
2.2 Hz, 1H; Ar), 7.07 (dd, J=2.2, 9.0 Hz, 1H; Ar), 5.18 (d, J=8.8 Hz,
1H; NH), 4.30 (d, J=6.0 Hz, 1H; ArCOCH CH CH), 3.31 (m, 2H; Ar-
COCH CH ), 2.30 (m, 1H; ArCOCH CH ), 2.11 (m, 1H; ArCOCH CH ),
1.47 (s, 9H; NHCOOC(CH ), 1.40 (s, 9H; COOC(CH ), 1.00 (s, 9H;
Si(CH C(CH ), 0.24 ppm (s, 6H; Si(CH
d=194.3 (ArCOCH ), 171.6 (COOtBu), 164.0 (NHCOO), 156.1 (Ar),
155.5 (Ar), 148.6 (Ar), 139.1 (Ar), 126.2 (Ar), 121.7 (Ar), 111.9 (Ar),
82.3 (COOC(CH ), 79.9 (NHCOOC(CH ), 53.6 (NHCHCOOtBu), 34.5
(ArCOCH CH ), 28.4 (COOC(CH ), 28.1 (NHCOOC(CH ), 27.2 (Ar-
), 25.7 (Si(CH C(CH ), 18.4 (Si(CH C(CH
C(CH
2
4
2
2
2
2
2
2
2
2
give 25 (10.4 mg, 90%) as white needlelike crystals. M.p. 155–
3
)
3
)
3 3
2
D
5
1
13
1
568C; [a] =À2.98 (c=1.00, CHCl ); H NMR (CD OD): d=7.91 (d,
)
3
2
3
)
3
) C(CH ) ); C NMR (CDCl ):
3 2 3 3 3
3
3
J=9.0 Hz, 1H; Ar), 7.36 (d, J=2.5 Hz, 1H; Ar), 7.09 (dd, J=2.5,
.0 Hz, 1H; Ar), 5.07 (dd, J=8.0, 10.3 Hz, 1H; CHCH O), 4.87–4.75
m, 2H; NCHCH O), 3.82 ppm (s, 3H; COOCH ); C NMR (CD OD):
2
9
(
2
1
3
)
3
)
3 3
2
3
3
3
d=172.3 (NCO), 162.8 (COOCH ), 159.2 (Ar), 152.3 (Ar), 148.0 (Ar),
2
2
3
)
3
)
3 3
3
1
6
39.2 (Ar), 126.0 (Ar), 118.6 (Ar), 107.2 (Ar), 72.4 (NCHCH O),
COCH
(Si(CH
CH
2
2
)
3
2
3
)
3
3
)
2
3
)
3
), À4.2 ppm
2
À1
À1
9.6 ppm (NCHCH O); IR (KBr): n˜ =3260, 1743, 1620 cm ; HRMS
3
)
2
3
)
3
); IR (film): n˜ =2930, 1717, 1710, 1703 cm ; HRMS
2
+
+
(
ESI): m/z calcd for C H N O S [M +H]: 278.0361; found:
(ESI): m/z calcd for C27H N O SSi [M +H]: 551.2611; found:
44 2 6
1
2
10
2
4
2
79.0442.
551.2599.
2
-Bromobenzo[d]thiazol-6-ol (18)
(R)-tert-Butyl-2-(tert-butoxycarbonylamino)-5-(6-hydroxyben-
zo[d]thiazol-2-yl)-5-oxopentanoate (24)
A 1.0m solution of BBr in CH Cl (4 mL) was added to a stirred so-
3
2
2
lution of 17 (555 mg, 2.28 mmol) in CH Cl (10 mL). The resulting
A solution of 1.0m TBAF (0.3 mL) in THF (1 mL), AcOH (0.057 mL),
2
2
mixture was stirred at room temperature for 2 h, diluted with cold
H O (30 mL), and extracted with CHCl . The organic layers were
and H O (0.018 mL) were added to a stirred solution of 23
(133 mg, 0.242 mmol) in THF (3 mL). The resulting mixture was
stirred at room temperature for 1 h, diluted with a saturated aque-
2
2
3
combined, washed with brine, dried over anhydrous Na SO , and
2
4
concentrated in vacuo. The residue was subjected to column chro-
ous solution of NH Cl (20 mL), and extracted with EtOAc. The or-
4
matography on silica gel (EtOAc/hexane, 1/3) to give 18 (521 mg,
ganic layers were combined, washed with brine, dried over anhy-
drous Na SO , and concentrated in vacuo. The residue was subject-
ed to column chromatography on silica gel (EtOAc/hexane, 1/1) to
1
9
7
5%) as a brown powder. M.p. 200–2028C; H NMR (CD OD): d=
3
2
4
.72 (d, J=8.8 Hz, 1H; Ar), 7.26 (d, J=2.4 Hz, 1H; Ar), 6.97 ppm
1
3
(
(
(
[
dd, J=2.4, 8.8 Hz, 1H; Ar); C NMR (CD OD): d=157.9 (Ar), 147.0
give 24 (100 mg, 95%) as a yellow powder. M.p. 180–1818C;
3
2
D
5
1
Ar), 139.8 (Ar), 136.2 (Ar), 123.8 (Ar), 117.4 (Ar), 107.2 ppm (Ar); IR
[a] =À17.2 (c=1.00, CH OH); H NMR (CD OD): d=7.89 (d, J=
3
3
À1
79
KBr): n˜ =3118, 1598 cm ; HRMS (ESI): m/z calcd for C H5 BrNOS
9.0 Hz, 1H; Ar), 7.30 (d, J=2.2 Hz, 1H; Ar), 7.09 (dd, J=2.2, 9.0 Hz,
1H; Ar), 5.35 (d, J=8.1 Hz, 1H; NH), 4.29 (d, J=5.6 Hz, 1H; Ar-
COCH CH CH), 3.29 (m, 2H; ArCOCH CH ), 2.28 (m, 1H; Ar-
7
+
M +H]: 229.9275; found: 229.9303.
2
2
2
2
COCH CH ), 2.10 (m, 1H; ArCOCH CH ), 1.46 (s, 9H; NHCOOC(CH ) ),
2
2
2
2
1
3 3
2-Bromo-6-(tert-butyldimethylsilyloxy)benzo[d]thiazole (19)
3
1
.43 ppm (s, 9H; COOC(CH ) );
C NMR (CD OD): d=194.1
3
3
3
Imidazole (1.41 g, 20.7 mmol) was added to a stirred solution of 18
(ArCOCH ), 171.6 (COOtBu), 162.9 (NHCOO), 157.1 (Ar), 156.0 (Ar),
2
(
794 mg, 3.45 mmol) and TBSCl (1.75 g, 11.6 mmol) in DMF (42 mL).
147.7 (Ar), 139.4 (Ar), 126.4 (Ar), 117.8 (Ar), 106.9 (Ar), 82.8
The resulting mixture was stirred at room temperature for 24 h, di-
(COOC(CH ) ), 80.6 (NHCOOC(CH ) ), 53.8 (NHCHCOOtBu), 34.6 (Ar-
3
3
3 3
luted with a saturated aqueous solution of NH Cl (50 mL), and ex-
COCH CH ), 28.5 (COOC(CH ) ), 28.1(NHCOOC(CH ) ), 27.1 ppm (Ar-
2 2 3 3 3 3
4
À1
tracted with CHCl . The organic layers were combined, washed
COCH CH ); IR (KBr): n˜ =3322, 1729, 1700 cm ; HRMS (ESI): m/z
2 2
3
+
with brine, dried over anhydrous Na SO , and concentrated in
calcd for C H N O S [M +H]: 437.1746; found: 437.1732.
21 29 2 6
2
4
vacuo. The residue was subjected to column chromatography on
silica gel (EtOAc/hexane, 1/3) to give 19 (1.21 g, quant.) as a brown
(
R)-5-(6-Hydroxybenzo[d]thiazol-2-yl)-3,4-dihydro-2H-pyr-
1
oil. H NMR (CDCl ): d=7.79 (d, J=8.8 Hz, 1H; Ar), 7.19 (d, J=
3
role-2-carboxylic acid (6)
2
.5 Hz, 1H; Ar), 6.94 (dd, J=2.5, 8.8 Hz, 1H; Ar), 0.97 (s, 9H;
1
3
Si(CH ) C(CH ) ), 0.20 ppm (s, 6H; Si(CH ) C(CH ) ); C NMR (CDCl3):
A solution of 24 (20.8 mg, 0.0476 mmol) in TFA (1 mL) was stirred
at room temperature for 24 h. The resulting mixture was concen-
trated in vacuo. The residue was subjected to reverse-phase prepa-
rative column chromatography on silica gel (H O/MeOH, 1/1) to
give 6 (10.3 mg, 82%) as yellow platelike crystals. M.p. 82–848C
3
2
3 3
3 2
3 3
d=154.1 (Ar), 147.3 (Ar), 138.4 (Ar), 135.9 (Ar), 123.2 (Ar), 120.3
Ar), 111.1 (Ar), 25.7 (Si(CH ) C(CH ) ), 18.2 (Si(CH ) C(CH ) ),
(
3
2
3 3
3 2
3 3
À1
À4.4 ppm (Si(CH ) C(CH ) ); IR (film): n˜ =1597 cm ; HRMS (ESI): m/
3
7
19
2
9
3 3
2
+
z calcd for C H BrNOSSi [M +H]: 344.0140; found: 344.0168.
1
3
2
D
5
1
(
(
9
dec); [a] =À26.5 (c=1.00, CH OH); H NMR ([D ]DMSO): d=7.93
3
6
d, J=9.2 Hz, 1H; Ar), 7.41 (d, J=2.5 Hz, 1H; Ar), 7.03 (dd, J=2.5,
.2 Hz, 1H; Ar), 4.87 (dd, J=6.7, 8.5 Hz, 1H; NCHCOOH), 3.17–3.08
m, 2H; ArCNCH CH ), 2.34 (m, 1H; ArCNCH CH ), 2.13 ppm (m, 1H;
(R)-tert-Butyl-2-(tert-butoxycarbonylamino)-5-[6-(tert-butyldi-
methylsilyloxy)benzo[d]thiazol-2-yl]-5-oxopentanoate (23)
(
2
3
2
2
2
1
A 2.7m solution of nBuLi in hexane (0.63 mL) was added to a stirred
solution of 19 (528 mg, 1.53 mmol) in dry THF (15.3 mL). The re-
ArCNCH CH ); C NMR (CD OD): d=175.2 (COOH), 175.0 (ArCN),
2 2 3
159.8 (Ar), 159.3 (Ar), 148.8 (Ar), 139.6 (Ar), 126.2 (Ar), 118.5 (Ar),
9336 ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2016, 22, 9330 – 9337
www.chemeurj.org

Full Paper
1
07.5 (Ar), 75.3 (NCHCOOH), 36.7 (ArCNCH CH ), 27.7 ppm
[4] Y. Ando, K. Niwa, N. Yamada, T. Enomoto, T. Irie, H. Kubota, Y. Ohmiya,
H. Akiyama, Nat. Photonics 2008, 2, 44.
[5] a) H. Fraga, Photochem. Photobiol. Sci. 2008, 7, 146; b) S. M. Marques,
J. C. G. Esteves de Silva, IUBMB Life 2009, 61, 6; c) S. Inouye, Cell. Mol.
Life Sci. 2010, 67, 387.
2
2
À1
(
ArCNCH CH ); IR (film): n˜ =3088, 2924, 1702, 1677, 1597 cm ;
2 2
+
HRMS (ESI): m/z calcd for C H N O S [M +H]: 263.0490; found:
2
1
2
11
2
3
63.0490.
[6] a) N. Thorne, J. Ingles, D. S. Auld, Chem. Biol. 2010, 17, 646; b) A. Roda,
M. Guardigli, E. Michelini, M. Mairasoli, TrAC Trends Anal. Chem. 2009,
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2
2
[
a stirred solution of 6 (10.0 mg, 0.0381 mmol) in MeOH (3 mL). The
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column chromatography on silica gel (EtOAc/hexane, 1/1) to give
1
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2
6 (3.20 mg, 33%) as a white powder. M.p. 237–2388C (dec);
25
1
[a] =À7.20 (c=1.00, CHCl ); H NMR ([D ]DMSO): d=7.93 (d, J=
D
3
6
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8
1
.8 Hz, 1H; Ar), 7.41 (d, J=2.5 Hz, 1H; Ar), 7.03 (dd, J=2.5, 8.8 Hz,
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3
NCHCOOCH ), 3.22–3.08 (m, 2H; ArCNCH CH ), 2.35 (m, 1H;
3
2
2
1
3
ArCNCH CH ), 2.14 ppm (m, 1H; ArCNCH CH ); C NMR (CD OD):
2
2
2
2
3
d=181.7 (COOCH ), 181.3 (ArCN), 168.7 (Ar), 166.6 (Ar), 156.2 (Ar),
3
3
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1
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(
(
NCHCOOCH ), 44.8 (ArCNCH CH ), 35.7 ppm (ArCNCH CH ); IR
3
2
2
À1
2
2
[
KBr): n˜ =3088, 1745, 1640 cm
; HRMS (ESI): m/z calcd for
+
C H N O S [M +H]: 277.0647; found: 277.0649.
13
13
2
3
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cemization to some extent was indicated by chiral HPLC; see Figure S5
in the Supporting Information. In 6, different optical rotation values (d
derivative: À26.5; l derivative: +19.8) and insufficient resolution of the
HPLC prevented assessment of the purity.
[
[
Acknowledgements
This study was partially supported by the Japan Student Ser-
vices Organization, JSPS KAKENHI Grant Number 24225001,
and Keio Leading-Edge Laboratory of Science and Technology.
Prof. Dr. Daniel Citterio gave us constructive comments and
warm encouragement.
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Keywords: biological
activity
·
firefly
luciferase
·
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luminescence
design
·
structure–activity relationships
·
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[
Received: January 20, 2016
Published online on May 24, 2016
1
Chem. Eur. J. 2016, 22, 9330 – 9337
www.chemeurj.org
9337
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim