Aminoluciferins as functional bioluminogenic substrates of firefly luciferase

Article abstract of DOI:10.1002/asia.201000873

Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD=the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.

Full text of DOI:10.1002/asia.201000873

FULL PAPERS
DOI: 10.1002/asia.201000873
Aminoluciferins as Functional Bioluminogenic Substrates of Firefly
Luciferase
Hideo Takakura,[a, b] Ryosuke Kojima,[a, b] Yasuteru Urano,[c] Takuya Terai,[a, b]
Kenjiro Hanaoka,[a, b] and Tetsuo Nagano*[a, b]
Abstract: Firefly luciferase is widely
used as a reporter gene in assays to
study gene expression, gene delivery,
and so on because of its extremely high
signal-to-noise ratio. The availability of
a range of bioluminogenic substrates
would greatly extend the applicability
of the luciferin–luciferase system.
Herein, we describe a design concept
for functional bioluminogenic sub-
strates based on the aminoluciferin
(AL) scaffold, together with a conven-
ient, high-yield method for synthesizing
N-alkylated ALs. We confirmed the
usefulness of ALs as bioluminogenic
substrates by synthesizing three probes.
The first was a conjugate of AL with
glutamate, Glu–AL. When Glu–AL,
the first membrane-impermeable biolu-
minogenic substrate of luciferases, was
applied to cells transfected with lucifer-
ase, luminescence was not observed;
that is, by using Glu–AL, we can distin-
guish between intracellular and extra-
cellular events. The second was Cy5–
AL, which consisted of Cy5, a near-in-
frared (NIR) cyanine fluorescent dye,
and AL, and emitted NIR light. When
Cy5–AL reacted with luciferase, lumi-
nescence derived from Cy5 was ob-
served as a result of bioluminescence
resonance energy transfer (BRET)
from AL to Cy5. The NIR emission
wavelength would allow a signal to be
observed from deeper tissues in biolu-
minescence in vivo imaging. The third
was biotin–DEVD–AL (DEVD=the
amino acid sequence Asp-Glu-Val-
Asp), which employed a caspase-3 sub-
strate peptide as a switch to control the
accessibility of the substrate to lucifer-
ase, and could detect the activity of
caspase-3 in a time-dependent manner.
This generalized design strategy should
be applicable to other proteases. Our
results indicate that the AL scaffold is
appropriate for a range of functional
luminophores and represents a useful
alternative substrate to luciferin.
Keywords: aminoluciferin · biolu-
minescent probes
· biosensors ·
firefly luciferase · luminescence
Introduction
Bioluminescence is employed in various highly sensitive in
vitro detection systems, as well as for in vivo imaging. One
of the most frequently used systems is the North American
firefly, Photinus pyralis, luciferin–luciferase system, which is
used as a reporter in biochemical assays, in cell cultures, and
recently in animal models to study gene expression, gene de-
livery, tumor progression, and so on.[1–4] Characteristic ad-
vantages of bioluminescence systems include a high quan-
tum yield (e.g., the recently reported value of Fbl =0.41)[5]
without excitation light and extremely high signal-to-noise
ratios compared with other imaging techniques, such as fluo-
rescence, even in vivo. Recently, bioluminescence systems
have been applied to detect other events, such as protein–
protein interactions and protease activity, by modification of
luciferase.[6–11] On the other hand, functional luciferin ana-
logues, the luminescence properties of which are changed
dramatically by specific activity, for instance, b-galactosi-
[a] H. Takakura, R. Kojima, T. Terai, Dr. K. Hanaoka,
Prof. Dr. T. Nagano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81) 3-5841-4855
E-mail: tlong@mol.f.u-tokyo.ac.jp
[b] H. Takakura, R. Kojima, T. Terai, Dr. K. Hanaoka,
Prof. Dr. T. Nagano
CREST Japan Science and Technology Agency
Honcho, Kawaguchi, Saitama 332-0012 (Japan)
[c] Prof. Dr. Y. Urano
Graduate School of Medicine
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000873.
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dase,[12] phosphatase,[13] glutathione S-transferase,[14] and lac-
tamase[15] activity, have also been reported as highly sensi-
tive indicators for target molecules. But, because the design
principle is based only on using luciferin 6’-ether or 6’-ester
derivatives, which have no emission prior to hydrolysis, the
range of available chemical modifications is restricted and
this approach is applicable to only a limited range of target
molecules. To further extend the applicability of the lucifer-
in–luciferase system, we focused on aminoluciferin (AL) as
a scaffold to develop functionalized bioluminescent sub-
strates. Herein, we present a novel design concept based on
AL modified with various functionalities at the 6’-position
(Scheme 1). As a first step, we developed an efficient syn-
strate (Glu–AL), a probe emitting NIR light (Cy5–AL), and
a probe for the detection of caspase-3 activity (biotin–
DEVD–AL; DEVD=the amino acid sequence Asp-Glu-
Val-Asp).
Results and Discussion
Improved Method for N-alkylation and Properties of AL
Derivatives
Firefly luciferase has a high specificity for firefly luciferin as
a substrate, therefore, it is difficult to introduce functionali-
ty, for example, 6’-ethers, 6’-esters, and 4-esters of luciferin
lack bioluminescence activity (Scheme 1). We focused on
AL as a scaffold because AL derivatives exhibit strong lumi-
nescence properties as substrates of luciferase.[16] N-Alkyla-
tion by the nucleophilic substitution reaction of the amino
group of 6-amino-2-cyanobenzothiazole (1) was reported by
Klaubert et al.,[16] but the reaction yields were quite low (5–
30%) owing to poor nucleophilicity of the amino group and
a long reaction time was required. Moreover, only monoal-
kylation could be achieved and not dialkylation. Thus, we
aimed to develop a more effective and convenient reaction.
We examined two kinds of reductive amination using the
corresponding aldehyde under acidic conditions: method A:
acid, H2SO4 (aq); reductant, NaBH4; solvent, tetrahydrofur-
an (THF); method B: acid, acetic acid; reductant,
NaBH3CN; solvent, acetonitrile. To examine whether these
reactions could be used as a general method for N-alkyla-
tion, we synthesized a series of derivatives with alkyl amino
moieties at the 6-position of 1, such as methyl (2a), dimethyl
(2b), ethyl (2c), diethyl (2d), benzyl (2e), phenylethyl (2 f),
phenylpropyl (2g), and phenylbutyl (2h) groups (Scheme 2).
The reaction yields were dramatically improved (39–91%,
see Table 1) compared with the previously reported method.
By using method A, low reaction yields were observed in
some cases, but by using method B the products were ob-
tained in good yields, although NaBH3CN was required. Ad-
ditionally, dialkylation of 1 can be performed in reasonable
yields (54% for dimethylation, 62% for diethylation). Then,
the corresponding AL derivatives (3a–3h) were obtained by
condensation with d-cysteine in aqueous methanol
(Scheme 2). Next, we investigated the luminescence proper-
ties of the products, especially the bioluminescence emission
maximum lmax. We observed luminescence with a longer-
wavelength emission peak than that of luciferin or AL for
all of the compounds synthesized (Table 1 and Figure S1 in
the Supporting Information). The kinetic parameters with
luciferase were measured and are summarized in Table 1.
The properties of the dialkylated AL derivatives were simi-
lar to those of the monoalkylated ALs.
Scheme 1. Chemical structures of firefly luciferin and AL derivatives de-
scribed herein.
thetic method for obtaining N-alkyl derivatives in higher
yield than those obtained with the method reported by
Klaubert et al.[16] We then confirmed the usefulness of AL
as a functional bioluminogenic scaffold by designing and
synthesizing the first membrane-impermeable luciferase sub-
Abstract in Japanese:
Design and Synthesis of Glu–AL
The above results provide a basis for the design and devel-
opment of functional bioluminescent substrates by using AL
as a scaffold. First, we tried to control the membrane perme-
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Scheme 2. Top: synthesis of N-alkylated AL. Reagents and conditions: Method A) 61 (2a), 54 (2b), 23 (2c), and 5% yield (for 2d); Method B) 91 (2c),
62 (2d); a) d-cysteine, K2CO3, H2O (pH 8)/MeOH, 78 (3a), 90 (3b), 83(3c), and 79% yield (3d). Bottom: synthesis of AL with a benzene ring. Reagents
AHCTUNGTRENNUNG
and conditions: Method A) 24 (2e), 39 (2 f), 57 (2g), and 53% yield (2h); Method B) 57% yield (2e), b) d-cysteine, K2CO3, H2O (pH 8)/MeOH, 42
(3e), 82 (3 f), 50 (3g), and 52% yield (3h). Details of methods A and B are given in the text.
plied to the plate, a signal was
clearly detected (Figure 1c and
Relative Vmax Figure S3b, c in the Supporting
Table 1. Reaction yields of N-alkylation of 2a–2h, along with lmax, Km, and relative Vmax data for AL deriva-
tives.
R1
R2
Reaction yield[a] [%]
Method A Method B
lmax
[nm][b]
Km
[mm][c]
Information). On the other
hand, in the case of Glu–AL,
AL
3a
3b
3c
3d
3e
3 f
H
Me
Me
Et
H
H
Me
H
Et
H
H
H
H
–
61
54
23
5
24
39
57
53
–
–
–
91
62
57
–
–
–
596
615
615
613
606
605
604
601
601
0.76Æ0.14
100[d]
0.071Æ0.006
0.059Æ0.002
0.033Æ0.003
0.12Æ0.01
25Æ1.2
16Æ1.0
22Æ1.4
16Æ0.9
1.6Æ0.09
7.3Æ0.08
39Æ3.4
2.2Æ0.09
the luminescence remained
almost at the background level;
however, strong luminescence
was observed in the lysate (Fig-
ure 1c and Figure S3a in the
Supporting Information). The
results indicate that Glu–AL is
a membrane-impermeable sub-
strate. Therefore, by using Glu–
AL, we can distinguish between
intra- and extracellular events.
For example, it may be possible
Et
benzyl
phenylethyl
phenylpropyl
phenylbutyl
0.041Æ0.003
0.081Æ0.005
0.42Æ0.04
3g
3h
0.34Æ0.03
[a] This represents reaction yields of N-alkylation of 2a–2h by reductive amination; method A: H2SO4,
NaBH4, THF; method B: AcOH, NaBH3CN, CH3CN. [b] Obtained from Figure S1 in the Supporting Informa-
tion. [c] Values of Km were determined from maximal light intensities after measurements to estimate the ini-
tial velocity.[17] [d] The Vmax value of AL was about 30% that of luciferin.
ability of substrates. Although luciferin has a carboxylic acid
moiety at the 4-position, it can permeate through the cell
membrane and, to the best of our knowledge, no mem-
brane-impermeable bioluminogenic substrates are available
(Figure 1a). We focused on the introduction of negatively
charged hydrophilic groups, such as carboxylic and sulfonic
acids (Figure 1a). This design strategy could not have been
applied to the luciferin scaffold because luciferin does not
lose luminescence activity upon the introduction of a func-
tional group at the 6’- or 4-positions. Thus, we prepared the
conjugate of 6-carboxymethylamino-2-cyanobenzothiazole
(4) with glutamate and the corresponding AL derivative
(Scheme 3), namely, Glu–AL, which has a total of three car-
boxylic acid moieties (Figure 1b) and investigated the lumi-
nescence properties and membrane permeability. The lumi-
nescence properties of Glu–AL were similar to those of N-
alkylated AL (Figure S2 and Table S1 in the Supporting In-
formation). We then cultured COS7 cells transfected with
luciferase in a 96-well plate. When AL or luciferin was ap-
to detect adenosine triphosphate (ATP) release sensitively[18]
or secretion of luciferase-fused proteins from cells after
some stimulation.[19]
Design and Synthesis of Cy5–AL
As mentioned in the Introduction, bioluminescence in vivo
imaging (BLI) can be used to sensitively monitor gene ex-
pression, gene delivery, and tumor progression. However,
for in vivo imaging, emission in the NIR region of l=650–
900 nm would be even better than bioluminescence from the
firefly luciferin–luciferase system because emission in this
region is subject to minimal interference from hemoglobin
absorption and offers good tissue penetration.[20,21] So far,
the longest wavelength maximum among luciferase variants
is about lmax =630 nm[22,23] and according to a recent report
on the fluorescence properties of oxyluciferin, this wave-
length appears to be the upper limit for firefly luciferin.[24]
To obtain NIR emission with luciferase, we designed an AL
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Aminoluciferins
from the substrate to the NIR
dye, thus affording NIR lumi-
nescence (Figure 2a). Based on
this approach, we synthesized a
bioluminogenic substrate con-
sisting of AL and Cy5, namely,
Cy5–AL
(Figure 2b
and
Scheme 4), and investigated
whether or not NIR lumines-
cence could be observed. First,
we confirmed that the absorb-
ance of Cy5 sufficiently over-
lapped with the luminescence
of AL (Figure 2c). As shown in
Figure 2d, the bioluminescence
of Cy5–AL was seen in the
NIR region (lmax =673 nm),
which was identical to the fluo-
rescence peak of Cy5, and the
luminescence of AL was not
observed at all, thereby suggest-
ing that highly efficient energy
transfer occurred. Thus, by uti-
lizing
this
luciferin-based
BRET strategy, it should be
possible to obtain multicolor lu-
minescence in the NIR region.
Although
a
luciferase-based
BRET strategy, in which NIR
dyes or quantum dots were
combined with luciferase, has
been reported,[25,26] it lacked
generality because the injection
of modified luciferase in vivo
Figure 1. a) Schematic illustration of cell membrane permeable and impermeable substrates. b) Chemical struc-
ture of Glu–AL. c) Light units (L.U. [a.u.]) of Glu–AL, AL, and luciferin. Red column: COS7 cells transfected
with luciferase, blue column: lysate of COS7 cells transfected with luciferase, green column: COS7 cells (not
transfected).
derivative linked with a NIR-emitting cyanine (Cy) dye.
When this substrate reacts with luciferase, bioluminescence
resonance energy transfer (BRET) is expected to occur
was required. Our strategy can be applied to conventional
systems and it should be possible to obtain signals from
deeper tissues in BLI.
Scheme 3. Synthesis of Glu–AL. Reagents and conditions: Method A) glyoxylic acid, 23%; Method B) glyoxylic acid, 90%; a) glutamic acid di-tert-butyl
ester, 1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophospate (HBTU), NEt3, N,N-dimethylforma-
mide (DMF), 67%; b) trifluoroacetic acid (TFA), CH2Cl2, 19%; c) d-cysteine, K2CO3, H2O (pH 8)/MeOH, quant. Details of methods A and B are given
in the text.
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T. Nagano et al.
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Information).[27] Therefore, a bulky substituent at the 6’-po-
sition of luciferin might block interaction with luciferase.
For this purpose, we chose avidin, which is a tetrameric pro-
tein containing four identical subunits, each of which binds
to biotin with high affinity and specificity. The dissociation
constant is KD ꢀ10À15 m, which makes avidin–biotin binding
one of the strongest known noncovalent interactions. We ex-
pected that AL with a biotin moiety, biotin–AL, would ex-
hibit bioluminescence like other AL derivatives, whereas
the complex between biotin–AL and avidin might not be-
cause avidin would block the access of luciferase to biotin–
AL (Figure 3a). Accordingly, we synthesized biotin–AL by
conjugating 4 with biotin, followed by condensation with d-
cysteine (Figure 3b and Scheme 5). We investigated the lu-
minescence of biotin–AL and the complex with avidin. As
shown in Figure 3c, bioluminescence was observed for
biotin–AL, but in the presence of avidin, the biolumines-
cence was lost. This result suggested that a huge molecule
located adjacent to the substrate could block interaction
with luciferase. We utilized this information to develop a
design strategy for a novel luminescence probe consisting of
three units: the first is a biotin site that can interact with
avidin to block access by luciferase, the second is a peptide
linker that contains a cleavage sequence for a target
enzyme, and the third is the substrate site (Figure 3b). As
proof of concept, we designed a caspase-3 probe with a
DEVD sequence, which was cleaved by caspase-3. We syn-
thesized biotin–DEVD–AL by means of solid-phase synthe-
sis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry
(Scheme S1 in the Supporting Information). The complex
with avidin did not show bioluminescence, but in the pres-
ence of caspase-3, time-dependent bioluminescence ap-
peared (Figure 3d). HPLC analysis of the reaction mixture
showed a single peak and the retention time of the reaction
product was identical to that of Lys–AL, which was formed
from biotin–DEVD–AL by caspase-3 (Figure 3e). The re-
sults indicated that biotin–DEVD–AL was degraded by cas-
pase-3 to Lys–AL with release of avidin, and Lys–AL emit-
ted bioluminescence. These findings demonstrate that lumi-
nescence can be switched on by controlling the accessibility
of the substrate to luciferase. This design strategy should be
applicable to not only aminopeptidases and exopeptidases,
but also to carboxypeptidases and endopeptidases. It is im-
portant to note that the enzymatic reaction of some exopep-
tidases is known to be influenced by the residue after the
scissile bond. For example, in the case of capase-3, charged
and bulky residues are poorly tolerated.[28] Conventionally,
an anilide group is required at the site, but with our strategy,
a wide range of amino acids can be used. Therefore, we en-
vision that this strategy could potentially be used in a wide
variety of applications for the detection of the activity of
various proteases, including for in vivo imaging.
Figure 2. a) Schematic illustration of NIR luminescent substrate based on
a BRET strategy. b) Chemical structure of Cy5–AL. c) Absorbance spec-
trum of Cy5–AL (c) and bioluminescence spectrum of AL (g).
d) Bioluminescence spectrum of Cy5–AL. I=normalized luminescence
intensity.
Design and Synthesis of a Substrate for Which
Bioluminescence is Controlled by Accessibility to
Luciferase
Finally, we focused on the X-ray crystal structure of the luci-
ferin–luciferase complex. It has been reported that the ben-
zothiazole moiety of firefly luciferin lies deep in the enzy-
matic active site of luciferase (Figure S4 in the Supporting
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Aminoluciferins
Scheme 4. Synthesis of Cy5–AL. Reagents and conditions: a) benzyloxycarbonyl (Cbz) chloride, NaHCO3, acetone/H2O, y. 94%; b) pyridinium chloro-
chromate (PCC), MgSO4, CH2Cl2, 16%; Method A) 1, 56%; c) TFA, CH2Cl2, 82%; d) Cy5-SE, NEt3, DMF, 27%; e) d-cysteine, K2CO3, H2O (pH 8)/
MeOH, 47%. Details of method A are given in the text.
Experimental Section
Conclusion
Materials
We developed an easy, quick, and high-yielding synthetic
In general, chemicals were of the best grade available, supplied by Wako
method for preparing N-alkylated ALs, which enabled us to
Pure Chemical, Tokyo Chemical Industries, and Aldrich Chemical Com-
efficiently synthesize AL derivatives with various functional
moieties. This makes it possible to design and develop a
wide range of novel functionalized AL-based bioluminogen-
ic substrates by chemical modification. We expected that
this approach would be very flexible, for example, allowing
modification of bioavailability, conjugation with biomole-
cules, control of luminescence activity or wavelength, and
easy targeting. To confirm the value of this methodology, we
introduced three types of functionality into bioluminogenic
substrates, demonstrating the feasibility of controlling distri-
bution by the use of a cell membrane impermeable probe,
adjusting luminescence emission to the NIR range by the
application of BRET, and detecting protease activity by in-
troducing an appropriate protease substrate sequence. These
results indicate that the AL scaffold should be suitable for a
range of functional luminophores, some of which would not
be feasible in a luciferin-based system, and thus, we propose
that AL represents a useful alternative substrate to luciferin.
pany, and were used without further purification. Special chemicals were
dimethyl sulfoxide (DMSO, fluorometric grade, Dojindo) and N,N-dime-
thylformamide (DMF) (fluorometric grade, Dojindo). Luciferase from
Photinus pyralis (molecular weight 120 kDa, E.C. 1.13.12.7) was pur-
chased from Sigma–Aldrich (Tokyo, Japan). Avidin from eggs (molecular
weight 66 kDa) was purchased from Pierce. NMR spectra were recorded
1
on a JNM-LA300 (JEOL) instrument at 300 MHz for H and at 75 MHz
for 13C nuclei. d values are given in ppm relative to tetramethylsilane.
Mass spectra (MS) were measured with JEOL JMS-T1000LC AccuTOF
(ESI) and JEOL SX-102A (EI) instruments. Bioluminescence spectra
were obtained on a Hitachi F4500 instrument. Bioluminescence kinetics
results were obtained on a Perkin–Elmer Envision instrument. The lumi-
nescence signal was detected by using a CCD camera attached to an
IVIS instrument (Xenogen). HPLC analyses were performed on an Inert-
sil ODS-3 (4.6ꢁ250 mm) analytical column and an Inertsil ODS-3 (10ꢁ
250 mm) semipreparative column using a HPLC system composed of a
pump (PU-2010, Jasco) and a detector (MD-2080, Jasco).
Synthesis and Characterization
Compound 1 was synthesized according to a reported method[29, 30] with
some modifications, as described in the Supporting Information
(Scheme S3). Cy5 was synthesized as reported.[31]
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Figure 3. a) Schematic illustration of the concept of blocking access to luciferase with a huge molecule. b) Chemical structures of biotin–AL, biotin–
DEVD–AL, and Lys–AL. c) Bioluminescence spectra of biotin–AL with (a) and without avidin (c). d) Bioluminescence signal of the complex be-
tween biotin–DEVD–AL and avidin in the presence and absence of caspase-3. e) HPLC analysis of biotin–DEVD–AL, Lys–AL, and the reaction mix-
ture of the complex between biotin–DEVD–AL and avidin with caspase-3. L.I.=luminescence intensity [a.u.], A=absorbance at 380 nm [a.u.].
General Procedure for N-Alkylation by Method A
stirred at room temperature. After 1 h, water and brine were added to
the reaction mixture, and the aqueous layer was separated and extracted
with ethyl acetate. The organic extract was dried over Na2SO4, filtered,
and evaporated. The product was purified by silica-gel column chroma-
tography (ethyl acetate/n-hexane) or by semipreparative HPLC (eluen-
t A: H2O/0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA).
The appropriate aldehyde (1.5 equiv for monoalkylation, 10 equiv for di-
alkylation) was dissolved in THF (20–40 mL). H2SO4 (180 mm, 1.5 equiv)
was added and the solution was stirred at room temperature for a few
minutes. A solution of 1 (50–120 mg, 1 equiv) and sodium borohydride
(1.5 equiv) in THF (40–60 mL) was added and the reaction mixture was
Scheme 5. Synthesis of biotin–AL. Reagents and conditions: a) biotin X cadaverine, HOBT, HBTU, NEt3, DMF; b) d-cysteine, K2CO3, H2O (pH 8)/
MeOH, 47% (2 steps).
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Aminoluciferins
General Procedure for N-Alkylation by Method B
13C NMR (75 MHz, CDCl3): d=148.7, 144.6, 138.6, 138.4, 129.5, 128.6,
126.6, 125.4, 117.0, 113.8, 99.8, 44.6, 34.9 ppm; HRMS (ESI+): m/z calcd
for C16H13N3S: 280.0908 [M+H]+; found: 280.0876.
The appropriate aldehyde (2 equiv for monoalkylation, 10 equiv for dia-
lkylation) was dissolved in CH3CN (10–20 mL). A solution of 1 (15~
30 mg, 1 equiv) and sodium cyanoborohydride (1.5 equiv) in CH3CN (10–
20 mL) was added, and then AcOH (1 mL) was added. The reaction mix-
ture was stirred at room temperature for 30 min. Methanol was added to
the reaction mixture to quench excess sodium cyanoborohydride. The
mixture was evaporated and the residue was extracted with ethyl acetate.
The organic extract was dried over Na2SO4, filtered, and evaporated. The
product was purified by silica-gel column chromatography (ethyl acetate/
n-hexane) or by semipreparative HPLC (eluent A: H2O/0.1% TFA,
eluent B: 80% CH3CN/20% H2O/0.1% TFA).
6-(3-Phenylpropylamino)-2-cyanobenzothiazole (2g)
Compound 2g was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:6) to give a yellow
solid (57%). 1H NMR (300 MHz, CDCl3): d=7.89 (d, J=9.5 Hz, 1H),
7.36–7.19 (m, 5H), 6.84–6.79 (m, 2H), 4.15 (brs, 1H), 3.22 (td, J=5.7,
7.2 Hz, 2H), 2.77 (t, J=7.4 Hz, 2H), 2.02 ppm (tt, J=7.2, 7.4 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=148.9, 144.5, 141.0, 138.7, 129.5, 128.5,
128.3, 126.1, 125.4, 117.0, 113.9, 99.6, 43.0, 33.2, 30.4 ppm; HRMS (ESI+):
m/z calcd for C17H15N3S: 294.1065 [M+H]+; found: 294.1091.
2-Cyano-6-methylaminobenzothiazole (2a)
6-(4-Phenylbutylamino)-2-cyanobenzothiazole (2h)
Compound 2a was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:6) to give a yellow
solid (61%). 1H NMR (300 MHz, CDCl3): d=7.92 (d, J=8.9 Hz, 1H),
6.92 (d, J=2.4 Hz, 1H), 6.88 (dd, J=2.4, 8.9 Hz, 1H), 4.27 (brs, 1H),
2.94 ppm (d, J=5.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=149.9,
144.6, 138.8, 129.5, 125.4, 116.8, 113.9, 99.2, 30.4 ppm; MS (EI+): m/z:
189 [M]+.
Compound 2h was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:6 to 1:3) to give a
yellow solid (53%). 1H NMR (300 MHz, CDCl3): d=7.89 (d, J=9.0 Hz,
1H), 7.32–7.16 (m, 5H), 6.88 (d, J=2.4 Hz, 1H), 6.84 (dd, J=2.4, 9.0 Hz,
1H), 3.20 (t, J=6.6 Hz, 2H), 2.69 (t, J=7.1 Hz, 2H), 1.84–1.66 ppm (m,
4H); 13C NMR (75 MHz, CDCl3): d=149.0, 144.5, 141.8, 138.8, 129.4,
128.3, 128.3, 125.9, 125.4, 116.9, 113.6, 99.5, 43.6, 35.4, 28.6, 28.5 ppm;
HRMS (ESI+): m/z calcd for C18H17N3S: 308.1221 [M+H]+; found:
308.1175.
2-Cyano-6-dimethylaminobenzothiazole (2b)
Compound 2b was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:6) to give a yellow
solid (54%). 1H NMR (300 MHz, CDCl3): d=7.98 (d, J=9.2 Hz, 1H),
7.06 (dd, J=2.6, 9.2 Hz, 1H), 7.01 (d, J=2.6 Hz, 1H), 3.10 ppm (s, 6H);
13C NMR (75 MHz, CDCl3): d=150.6, 143.8, 138.6, 129.4, 125.1, 114.8,
114.0, 100.7, 40.6 ppm; MS (EI+): m/z: 203 [M]+.
General Procedure for Cyclization of 2-Cyanobenzothiazoles
d-Cysteine hydrochloride monohydrate (3 equiv) was dissolved in water
(10 mL) under argon bubbling and the pH of the solution was adjusted
to 8 with potassium carbonate (0.5m). The appropriate 2-cyanobenzothia-
zole compound (1 equiv) was dissolved in methanol (10–15 mL) with
argon bubbling and the d-cysteine solution was added. The mixture was
then stirred at room temperature under an argon atmosphere in the dark
for 1–2 h. The reaction mixture was acidified to about pH 4 with hydro-
chloric acid and the methanol was evaporated. The residue was extracted
with ethyl acetate, and the organic solution was washed with brine, dried
over Na2SO4, filtered, and evaporated. The product was purified by semi-
preparative HPLC (eluent A: H2O/0.1% TFA, eluent B: 80% CH3CN/
20% H2O/0.1% TFA), or recrystallized from ethanol/n-hexane.
2-Cyano-6-ethylaminobenzothiazole (2c)
Compound 2c was prepared by Method A or B and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:4) to give a yellow
solid (23% for Method A; 91% for Method B). 1H NMR (300 MHz,
CDCl3): d=7.91 (d, J=9.0 Hz, 1H), 6.92 (d, J=2.2 Hz, 1H), 6.87 (dd,
J=2.2, 9.0 Hz, 1H), 4.13 (brs, 1H), 3.30–3.18 (m, 2H), 1.33 ppm (t, J=
7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=149.0, 144.6, 138.8, 129.6,
125.5, 116.9, 113.8, 99.6, 38.3, 14.4 ppm; HRMS (ESI+): m/z calcd for
C10H9N3S: 204.0595 [M+H]+; found: 204.0555.
d-(À)-2-(6’-Methylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic
acid (3a)
Compound 3a was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=80:20 to
20:80 (20 min)) and recrystallized from ethanol/n-hexane to give a red
solid (78%). M.p. 132–1348C (dec); 1H NMR (300 MHz, CD3OD): d=
7.75 (d, J=9.0 Hz, 1H), 6.99 (d, J=2.3 Hz, 1H), 6.88 (dd, J=2.3, 9.0 Hz,
1H), 5.35 (t, J=9.1 Hz, 1H), 3.78–3.67 (m, 2H), 2.84 ppm (s, 3H);
13C NMR (75 MHz, CD3OD): d=173.3, 167.9, 157.0, 148.5, 147.9, 140.0,
125.7, 118.0, 104.3, 79.2, 35.8, 32.0 ppm; HRMS (ESI+): m/z calcd for
C12H11N3O2S2: 294.0371 [M+H]+; found: 294.0340; elemental analysis
calcd (%) for C12H11N3O2S2·0.25H2O: C 48.4, H 3.9, N 14.1; found: C
48.5, H 4.2, N 13.7.
2-Cyano-6-diethylaminobenzothiazole (2d)
Compound 2d was prepared by Method A or B and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:5) to give a yellow
solid (5% for Method A; 62% for Method B). 1H NMR (300 MHz,
CDCl3): d=7.95 (d, J=9.2 Hz, 1H), 7.01 (dd, J=2.6, 9.2 Hz, 1H), 6.97
(d, J=2.6 Hz, 1H), 3.46 (q, J=7.1 Hz, 4H), 1.24 ppm (t, J=7.1 Hz, 6H);
13C NMR (75 MHz, CDCl3): d=148.4, 143.3, 139.1, 128.9, 125.5, 114.4,
114.0, 99.9, 45.0, 12.5 ppm; HRMS (ESI+): m/z calcd for C12H13N3S:
232.0908 [M+H]+; found: 232.0872.
6-Benzylamino-2-cyanobenzothiazole (2e)
d-(À)-2-(6’-Dimethylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic
acid (3b)
Compound 2e was prepared by Method A or B and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:4) and semiprepara-
tive HPLC (eluent A: H2O/0.1% TFA, eluent B: 80% CH3CN/20%
H2O/0.1% TFA, A/B=50:50 to 0:100 (10 min)) to give a yellow solid
(57%). 1H NMR (300 MHz, CDCl3): d=7.93 (d, J=8.7 Hz, 1H), 7.39–
7.30 (m, 5H), 6.97–6.91 (m, 2H), 4.61 (brs, 1H), 4.43 ppm (d, J=5.6 Hz,
2H); 13C NMR (75 MHz, CDCl3): d=148.7, 144.8, 138.6, 137.7, 129.9,
128.9, 127.7, 127.3, 125.5, 117.0, 113.8, 100.2, 48.0 ppm; HRMS (ESI+):
m/z calcd for C15H11N3S: 266.0752 [M+H]+; found: 266.0759.
Compound 3b was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=80:20 to
20:80 (20 min)) and recrystallized from ethanol/n-hexane to give a red
solid (90%). M.p. 177–1788C (dec); 1H NMR (300 MHz, [D6]DMSO):
d=13.22 (brs, 1H), 7.98 (d, J=9.3 Hz, 1H), 7.38 (d, J=2.5 Hz, 1H), 7.14
(dd, J=2.5, 9.3 Hz, 1H), 5.44 (dd, J=8.3, 9.6 Hz, 1H), 3.81 (dd, J=9.6,
11.4 Hz, 1H), 3.70 (dd, J=8.3, 11.4 Hz, 1H), 3.10 ppm (s, 6H); 13C NMR
(75 MHz, [D6]acetone): d=171.5, 166.2, 156.0, 150,9, 146.1, 139.5, 125.3,
115.0, 103.0, 79.2, 41.0, 35.3 ppm; HRMS (ESI+): m/z calcd for
C13H13N3O2S2: 308.0527 [M+H]+; found: 308.0481; elemental analysis
calcd (%) for C13H13N3O2S2: C 50.8, H 4.3, N 13.7; found: C 50.6, H 4.4,
N 13.4.
6-Phenethylamino-2-cyanobenzothiazole (2 f)
Compound 2 f was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:6) to give a yellow
solid (39%). 1H NMR (300 MHz, CDCl3): d=7.91 (d, J=9.0 Hz, 1H),
7.38–7.21 (m, 5H), 6.95 (d, J=2.4 Hz, 1H), 6.85 (dd, J=2.4, 9.0 Hz, 1H),
4.22 (brs, 1H), 3.49 (td, J=5.9, 6.9 Hz, 2H), 2.98 ppm (t, J=6.9 Hz, 2H);
Chem. Asian J. 2011, 6, 1800 – 1810
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1807

T. Nagano et al.
FULL PAPERS
d-(À)-2-(6’-Ethylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic acid
(3c)
d-(À)-2-[6’-(4-Phenylbutylamino)-2’-benzothiazolyl]-D2-thiazoline-4-
carboxylic acid (3h)
Compound 3c was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=80:20 to
20:80 (20 min)) and recrystallized from ethanol/n-hexane to give a red
solid (83%). M.p. 102–1048C; 1H NMR (300 MHz, CD3CN): d=7.82 (d,
J=9.0 Hz, 1H), 7.16 (d, J=2.2 Hz, 1H), 6.96 (dd, J=2.2, 9.0 Hz, 1H),
5.35 (t, J=9.2 Hz, 1H), 3.75–3.62 (m, 2H), 3.21 (q, J=7.2 Hz, 2H),
1.24 ppm (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CD3OD): d=173.3,
167.7, 158.0, 148.6, 146.0, 139.8, 125.8, 118.7, 106.3, 79.3, 42.0, 35.9,
13.6 ppm; HRMS (ESI+): m/z calcd for C13H13N3O2S2: 308.0527
[M+H]+; found: 308.0483; elemental analysis calcd (%) for
C13H13N3O2S2·0.25H2O·0.25EtOH: C 50.1, H 4.7, N 13.0; found: C 50.2,
H 4.6, N 12.7.
Compound 3h was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to
0:100 (20 min)) to give a red solid (82%). H NMR (300 MHz, CD3OD):
d=7.85 (d, J=9.0 Hz, 1H), 7.28–7.12 (m, 6H), 7.05 (dd, J=2.4, 9.0 Hz,
1H), 5.37 (t, J=9.1 Hz, 1H), 3.80–3.71 (m, 2H), 3.24 (t, J=7.0 Hz, 2H),
2.67 (t, J=7.2 Hz, 2H), 1.82–1.66 ppm (m, 4H); 13C NMR (75 MHz,
[D6]acetone): d=173.3, 167.8, 157.8, 148.6, 146.0, 143.2, 139.8, 129.4,
129.3, 126.8, 125.8, 118.8, 106.3, 79.2, 47.1, 36.4, 35.9, 29.7, 28.6 ppm;
HRMS (ESI+): m/z calcd for C21H21N3O2S2: 412.1153 [M+H]+; found:
412.1113.
1
6-Carboxymethylamino-2-cyanobenzothiazole (4)
Compound 4 was prepared by Method A or B and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:3 to 1:1) and semi-
preparative HPLC (eluent A: H2O/0.1% TFA, eluent B: 80% CH3CN/
20% H2O/0.1% TFA, A/B=80:20 to 20:80 (20 min)) to give a yellow
solid (23% for Method A; 90% for Method B). 1H NMR (300 MHz,
CD3OD): d=7.87 (d, 1H, J=9.9 Hz), 7.08–7.04 (m, 2H), 3.99 ppm (s,
2H); 13C NMR (75 MHz, [D6]acetone): d=171.7, 150.2, 145.5, 139.6,
130.3, 126.0, 118.2, 114.6, 101.2, 45.2 ppm; HRMS (ESI+): m/z calcd for
C10H7N3O2S: 234.0337 [M+H]+; found: 234.0300.
d-(À)-2-(6’-Diethylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic
acid (3d)
Compound 3d was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=80:20 to
1
20:80 (20 min)) to give a red solid (79%). H NMR (300 MHz, CD3OD):
d=8.09 (d, J=9.0 Hz, 1H), 7.94 (d, J=2.4 Hz, 1H), 7.47 (dd, J=2.4,
9.0 Hz, 1H), 3.74–3.64 (m, 2H), 3.59 (t, J=7.2 Hz, 4H), 1.08 ppm (t, J=
7.2 Hz, 6H); 13C NMR (75 MHz, CD3OD): d=173.5, 167.7, 155.7, 148.7,
145.7, 140.4, 125.5, 115.1, 102.8, 79.4, 46.1, 35.9, 12.8 ppm; HRMS (ESI+):
m/z calcd for C15H17N3O2S2: 336.0840 [M+H]+; found: 336.0812.
2-Cyano-6-[2-(1,3-di-tert-butoxycarbonylpropylamino)-2-
oxoethylamino]benzothiazole (5)
d-(À)-2-(6’-Benzylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic
acid (3e)
1-Hydroxybenzotriazole (HOBT; 23 mg, 150 mmol) and 2-(1H-benzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophospate (HBTU; 60 mg,
160 mmol) were added to a solution of 1 (11 mg, 48 mmol) in DMF
(2 mL) cooled on an ice bath. After a few minutes, glutamic acid di-tert-
butyl ester (38 mg, 130 mmol) and ten drops of NEt3 were added, then
the solution was stirred for 1 h at room temperature. Water was added
and the reaction mixture was extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4, filtered, and evaporated.
The product was purified by silica-gel column chromatography (ethyl
acetate/n-hexane=1:3) and semipreparative HPLC (eluent A: H2O/0.1%
TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to 0:100
(10 min)) to give a yellow solid (15 mg, 67%). 1H NMR (300 MHz,
CDCl3): d=7.98 (d, 1H, J=9.7 Hz), 7.10–7.04 (m, 1H), 7.02–6.95 (m,
2H), 4.58–4.52 (m, 1H), 3.93 (s, 2H), 2.30–2.20 (m, 2H), 2.18–2.05 (m,
1H), 1.95–1.82 (m, 1H), 1.43 (s, 9H), 1.41 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3): d=172.3, 171.2, 169.1, 147.8, 145.7, 138.4, 131.2,
125.8, 117.2, 113.6, 101.2, 82.7, 81.1, 52.3, 47.8, 31.4, 28.0, 28.0, 27.3 ppm;
HRMS (ESI+): m/z calcd for C23H30N4O5S: 497.1835 [M+Na]+; found:
497.1822.
Compound 3e was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to
0:100 (20 min)) and recrystallized from ethanol/n-hexane to give a red
solid (42%). M.p. 108–1098C; 1H NMR (300 MHz, CD3OD): d=7.77 (d,
J=8.9 Hz, 1H), 7.41–7.20 (m, 5H), 7.02 (d, J=2.2 Hz, 1H), 6.97 (dd, J=
2.2, 8.9 Hz, 1H), 5.34 (t, J=9.1 Hz, 1H), 4.41 (s, 2H), 3.78–3.66 ppm (m,
2H); 13C NMR (75 MHz, CD3OD): d=173.3, 167.9, 155.8, 149.3, 146.7,
140.0, 139.9, 129.6, 128.5, 128.2, 125.5, 117.5, 102.9, 79.1, 48.9, 35.7 ppm;
HRMS (ESI+): m/z calcd for C18H15N3O2S2: 370.0684 [M+H]+; found:
370.0683; elemental analysis calcd (%) for C18H15N3O2S2·0.5H2O: C 57.1,
H 4.3, N 11.1; found: C 57.1, H 4.5, N 10.7.
d-(À)-2-(6’-Phenethylamino-2’-benzothiazolyl)-D2-thiazoline-4-carboxylic
acid (3 f)
Compound 3 f was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to
1
0:100 (20 min)) to give a red solid (82%). H NMR (300 MHz, CD3OD):
d=7.79 (d, J=9.0 Hz, 1H), 7.31–7.19 (m, 5H), 7.13 (d, J=2.3 Hz, 1H),
6.94 (dd, J=2.3, 9.0 Hz, 1H), 5.36 (t, J=9.0 Hz, 1H), 3.79–3.68 (m, 2H),
3.45 (t, J=7.3 Hz, 2H), 2.94 ppm (t, J=7.3 Hz, 2H); 13C NMR (75 MHz,
CD3OD): d=173.3, 167.9, 156.0, 148.6, 147.0, 140.5, 140.1, 129.8, 129.5,
127.4, 125.6, 117.6, 103.2, 79.0, 47.0, 35.9, 35.8 ppm; HRMS (ESI+): m/z
calcd for C19H17N3O2S2: 384.0840 [M+H]+; found: 384.0816.
2-Cyano-6-[2-(1,3-dicarboxypropylamino)-2-oxoethylamino]benzothiazole
(6)
TFA (2 mL) was added to a solution of 5 (15 mg, 32 mmol) in CH2Cl2
(2 mL) and the reaction mixture was stirred for 2 h at room temperature.
The reaction mixture was evaporated with toluene to give the crude
product. The product was purified by semipreparative HPLC (eluent A:
H2O/0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=
80:20 to 20:80 (20 min)) to give a yellow solid (2 mg, 19%). 1H NMR
(300 MHz, CD3OD): d=7.80 (d, J=9.7 Hz, 1H), 7.00–6.95 (m, 2H),
4.44–4.35 (m, 1H), 3.84 (s, 2H), 2.21 (t, J=7.7 Hz, 2H), 2.15–2.05 (m,
1H), 1.90–1.76 ppm (m, 1H); HRMS (ESIÀ): m/z calcd for C15H14N4O5S:
361.0607 [MÀH]À; found: 361.0595.
d-(À)-2-[6’-(3-Phenylpropylamino)-2’-benzothiazolyl]-D2-thiazoline-4-
carboxylic acid (3g)
Compound 3g was purified by semipreparative HPLC (eluent A: H2O/
0.1% TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to
0:100 (20 min)) and recrystallized from ethanol/n-hexane to give a red
solid (42%). M.p. 128–1298C; 1H NMR (300 MHz, CD3OD): d=7.71 (d,
J=9.0 Hz, 1H), 7.22–7.12 (m, 5H), 6.98 (d, J=2.4 Hz, 1H), 6.87 (dd, J=
2.4, 9.0 Hz, 1H), 5.27 (t, J=9.0 Hz, 1H), 3.70–3.58 (m, 2H), 3.11 (t, J=
7.3 Hz, 2H), 2.65 (t, J=7.5 Hz, 2H), 1.88 ppm (tt, J=7.3, 7.5 Hz, 2H);
13C NMR (75 MHz, CD3OD): d=173.3, 167.8, 156.6, 147.4, 142.8, 140.0,
129.4, 127.0, 125.6, 118.0, 104.0, 101.9, 79.1, 45.4, 35.8, 34.1, 31.2 ppm;
HRMS (ESI+): m/z calcd for C20H19N3O2S2: 398.0997 [M+H]+; found:
398.0959; elemental analysis calcd (%) for C20H19N3O2S2: C 60.4, H 4.8,
N 10.6; found: C 60.4, H 4.9, N 10.4.
d-(À)-2-{6’-[2-(1,3-dicarboxypropylamino)-2-oxoethylamino]-2’-
benzothiazolyl}-D2-thiazoline-4-carboxylic acid (Glu–AL)
Glu–AL was purified by semipreparative HPLC (eluent A: H2O/0.1%
TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to 0:100
(20 min)) to give a red solid (quant). 1H NMR (300 MHz, CD3OD): d=
7.81 (d, J=9.0 Hz, 1H), 7.04 (d, J=2.2 Hz, 1H), 6.96 (dd, J=2.2, 9.0 Hz,
1H), 5.36 (t, J=9.1 Hz, 1H), 4.52–4.48 (m, 1H), 3.91 (s, 2H), 3.78–3.68
(m, 2H), 2.30 (t, J=6.6 Hz, 2H), 2.25–2.16 (m, 1H), 2.00–1.90 ppm (m,
1H); 13C NMR (100 MHz, CD3OD): d=176.3, 174.6, 173.4, 173.3, 167.7,
1808
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1800 – 1810

Aminoluciferins
156.5, 149.6, 147.0, 139.9, 125.5, 117.1, 102.5, 79.4, 52.9, 48.3, 35.8, 31.0,
27.7 ppm; HRMS (ESI+): m/z calcd for C18H18N4O7S2: 467.0695
[M+H]+; found: 467.0722.
rified with semipreparative HPLC (eluent A: H2O/0.1% TFA, eluent B:
80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to 0:100 (10 min)) to
give a blue solid (18 mg, 27% as a zwitterion). 1H NMR (300 MHz,
CD3OD): d=8.24 (t, J=13.0 Hz, 2H), 8.15 (brm, 1H), 7.77 (d, J=
8.6 Hz, 1H), 7.46–7.20 (m, 8H), 6.98–6.91 (m, 2H), 6.60 (t, J=13.0 Hz,
1H), 6.31 (d, J=13.0 Hz, 2H), 4.39–4.30 (m, 4H), 3.13–3.04 (m, 4H),
2.78 (t, J=7.1 Hz, 2H), 2.64 (t, J=6.3 Hz, 2H), 1.75–1.66 (m, 16H),
1.64–1.51 ppm (m, 4H); HRMS (ESI+): m/z calcd for C45H50N6O3S:
755.3743 [M]+; found: 755.3748.
6-(Benzyloxycarbonylamino)-1-hexanol (8)
Compound 7 (2.89 g, 25 mmol) was dissolved in water (120 mL) and ace-
tone (240 mL), and NaHCO3 (2.3 g, 28 mmol) was added to the solution.
Cbz chloride (4.71 g, 28 mmol) was slowly added over 5 min and the reac-
tion mixture was stirred at room temperature. After 24 h, acetone was
evaporated and the precipitate was collected by filtration, washed with
water, and dried under high vacuum to afford a white solid without pu-
rification (5.84 g, 94%). 1H NMR (300 MHz, CDCl3): d=7.38–7.31 (m,
5H), 5.10 (s, 2H), 4.75 (brs, 1H), 3.63 (td, J=5.9, 6.4 Hz, 2H), 3.20 (td,
J=6.1, 6.8 Hz, 2H), 1.62–1.50 (m, 4H), 1.45–1.27 ppm (m, 5H);
13C NMR (75 MHz, CDCl3): d=156.4, 136.5, 128.5, 128.0, 66.5, 62.6, 40.8,
32.5, 29.9, 26.3, 25.2 ppm; HRMS (ESI+): m/z calcd for C14H21NO3:
274.1419 [m+Na]+; found: 274.1394.
Synthesis of Cy5–AL
Cy5–AL was purified by semipreparative HPLC (eluent A: H2O/0.1%
TFA, eluent B: 80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to 0:100
(20 min)) to give a red solid (47% as a zwitterion). 1H NMR (300 MHz,
CD3OD): d=8.21 (d, J=13.0 Hz, 2H), 7.71 (d, J=9.0 Hz, 1H), 7.45–7.19
(m, 8H), 6.95 (d, J=2.3 Hz, 1H), 6.87 (dd, J=2.3, 9.0 Hz, 1H), 6.58 (t,
J=13.0 Hz, 1H), 6.30 (d, J=13.0 Hz, 2H), 5.35 (t, J=9.1 Hz, 1H), 4.39–
4.31 (m, 4H), 3.76–3.66 (m, 2H), 3.14–3.04 (m, 4H), 2.77 (t, J=7.1 Hz,
2H), 2.63 (t, J=6.2 Hz, 2H), 1.70–1.65 (m, 12H), 1.63–1.52 (m, 2H),
1.42–1.30 (m, 4H), 1.29–1.19 ppm (m, 2H); 13C NMR (75 MHz,
CD3OD): d=174.9, 174.9, 173.8, 173.1, 172.1, 167.7, 155.8, 155.2, 150.5,
146.1, 143.2, 143.1, 142.5, 142.5, 140.2, 129.7, 127.1, 126.4, 126.4, 125.4,
123.4, 123.3, 117.0, 112.1, 109.1, 104.9, 104.6, 101.3, 79.3, 50.6, 44.5, 41.8,
41.0, 40.6, 35.8, 35.0, 32.6, 30.2, 29.8, 28.0, 27.9, 27.8, 27.6 ppm; HRMS
(ESI+): m/z calcd for C48H54N6O5S2: 859.3675 [M]+; found: 859.3671.
6-(Benzyloxycarbonylamino)-1-hexanal (9)
Pyridinium chlorochromate (PCC; 7.45 g, 35 mmol) was dissolved in
CH2Cl2 (120 mL) and MgSO4 (3.1 g) was added. A solution of 8 (5.77 g,
23 mmol) in CH2Cl2 (50 mL) was also added and the reaction mixture
was stirred at room temperature for 4 h. The mixture was filtered, the
filter was washed with CH2Cl2, and the filtrate was evaporated. The resi-
due was purified by silica-gel column chromatography (ethyl acetate/n-
hexane=1:2) to give a colorless oil (0.88 g, 16%). 1H NMR (300 MHz,
CDCl3): d=9.76 (s, 1H), 7.40–7.28 (m, 5H), 5.10 (s, 2H), 4.75 (brs, 1H),
3.20 (td, J=6.4, 6.6 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H), 1.73–1.29 ppm (m,
6H); 13C NMR (75 MHz, CDCl3): d=202.4, 156.3, 136.4, 128.3, 127.9,
66.3, 43.5, 40.6, 29.5, 26.0, 21.4 ppm; HRMS (ESI+): m/z calcd for
C14H19NO3: 272.1263 [M+Na]+; found: 272.1235.
Synthesis of 13
HOBT (5 mg, 30 mmol) and HBTU (12 mg, 30 mmol) were added to a so-
lution of 4 (7 mg, 30 mmol) in DMF (2 mL) cooled in an ice bath. After a
few minutes, biotin X cadaverine (10 mg, 20 mmol) and a few drops of
NEt3 were added and the solution was stirred for 1 h at room tempera-
ture. Water was added, and the reaction mixture was evaporated. The
product was used for the next reaction without further purification.
HRMS (ESIÀ): m/z calcd for C31H44N8O4S2: 655.2849 [MÀH]À; found:
655.2897.
6-[6-(Benzyloxycarbonylamino)hexylamino]-2-cyanobenzothiazole (10)
Compound 10 was prepared by Method A and purified by silica-gel
column chromatography (ethyl acetate/n-hexane=1:5 to 1:2) to give a
yellow solid (56%). 1H NMR (300 MHz, CDCl3): d=7.90 (d, J=8.9 Hz,
1H), 7.38–7.31 (m, 5H), 6.90 (d, J=2.3 Hz, 1H), 6.87 (dd, J=2.3, 8.9 Hz,
1H), 5.10 (s, 2H), 4.74 (brs, 1H), 4.25 (brs, 1H), 3.28–3.14 (m, 4H), 1.67
(q, J=7.0 Hz, 2H), 1.59–1.34 ppm (m, 6H); 13C NMR (75 MHz, CDCl3):
d=156.4, 149.1, 144.4, 138.7, 136.5, 129.2, 128.4, 128.0, 128.0, 125.3, 116.9,
113.9, 99.4, 66.5, 43.4, 40.7, 29.8, 28.8, 26.4, 26.2 ppm; HRMS (ESI+): m/z
calcd for C22H24N4O2S: 431.1518 [M+Na]+; found: 431.1510.
Synthesis of Biotin–AL
Purified by semipreparative HPLC (eluent A: H2O/0.1% TFA, eluent B:
80% CH3CN/20% H2O/0.1% TFA, A/B=50:50 to 0:100 (20 min)) to
give
a
red solid (47% (2 steps)). HRMS (ESI+): m/z calcd for
C34H48N8O6S3: 783.2757 [M+Na]+; found: 783.2787.
Measurement of Bioluminescence Kinetics (Figure 1c)
6-(6-Aminohexylamino)-2-cyanobenzothiazole (11)
The COS7 cell line was purchased from RIKEN BioResource Center
and maintained in Dulbeccoꢂs modified eagle medium (DMEM; Sigma,
cat. no.: D5796) containing 10% fetal bovine serum (FBS; Gibco, cat.
no.: 10082) and 1% penicillin/streptomycin (Gibco, cat. no.: 15140). Luci-
ferase plasmid was kindly provided by Professor Ozawa (Department of
Chemistry, School of Science, The University of Tokyo). COS7 cell line
was transfected with the plasmid by using Lipofectamine 2000 (Invitro-
gen), and after 24 h the assay was carried out. COS7 cells transfected
with luciferase were cultured on 96-well plates. Measurements were con-
ducted with a plate reader following injection of 100 mm (final) substrate.
In the lysate assay, the measurements were conducted after cell lysis
(Glo lysis, Promega) for 5 min. The light unit is defined as luminescence
accumulation for 10 min from injection of substrates.
Compound 10 (70 mg, 170 mmol) was dissolved in CH2Cl2 (5 mL) and
TFA (20 mL) was added to the solution. The reaction mixture was stirred
at room temperature for 16 h, then evaporated, and the residue was dis-
solved in 1n HCl (aq). The aqueous solution was washed with CH2Cl2
and extracted with ethyl acetate after neutralization. The organic layer
was washed with brine, dried over Na2SO4, evaporated, and dried under
high vacuum to afford a red solid (38 mg, 82%). 1H NMR (300 MHz,
CD3OD): d=7.81 (d, J=9.0 Hz, 1H), 7.04 (d, J=2.3 Hz, 1H), 6.99 (dd,
J=2.3, 9.0 Hz, 1H), 3.18 (t, J=7.0 Hz, 2H), 2.92 (t, J=7.6 Hz, 2H),
1.73–1.61 (m, 4H), 1.54–1.42 ppm (m, 4H); 13C NMR (75 MHz,
CD3OD): d=151.7, 145.1, 140.4, 129.7, 125.9, 118.4, 114.9, 100.1, 44.2,
40.7, 29.6, 28.5, 27.7, 27.3 ppm; HRMS (ESI+): m/z calcd for C14H18N4S:
275.1330 [M+H]+; found: 275.1293.
Measurement of Absorbance Spectrum (Figure 2c)
Synthesis of 12
3 mm Cy5–AL was dissolved in 30 mm 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES) buffer (pH 7.7) and the absorbance spec-
trum was measured.
Cy5 (34 mg, 68 mmol) was dissolved in DMF (5 mL), and 1,3-dicyclohex-
ylcarbodiimide (DCC; 18 mg, 88 mmol) and N-hydroxysuccinimide
(NHS; 10 mg, 89 mmol) were added to the solution. The reaction mixture
was stirred at room temperature and the progress of the reaction was
monitored by HPLC (eluent A: H2O/0.1% TFA, eluent B: 80% CH3CN/
20% H2O/0.1% TFA, A/B=50:50 to 0:100 (10 min)). After 5 h (Cy5/
Cy5-SE/Cy5-diSE=0.8:1:0.2; SE=succinimidyl ester), a solution of 11 in
DMF (2 mL) and NEt3 (12 mL) was added to the reaction mixture, and
stirring was continued at room temperature for 2 h. The mixture was pu-
Measurement of Bioluminescence Spectra (Figure 2d)
The reaction cocktail (30 mm HEPES buffer, 5 mm MgSO4, 2.6 mm ATP,
3.5 mm dithiothreitol (DTT), 25 mgmLÀ1 luciferase, pH 7.7) was prepared.
Measurements were taken immediately after the addition of 5 mm (final)
Cy5–AL at 258C. The solution contained up to 0.5% (v/v) DMF as a co-
Chem. Asian J. 2011, 6, 1800 – 1810
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1809

T. Nagano et al.
FULL PAPERS
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The reaction cocktail (30 mm HEPES buffer, 5 mm MgSO4, 2.6 mm ATP,
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Caspase-3 Assay and Bioluminescence Assay (Figure 3d)
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reaction mixture and then the bioluminescence reaction was initiated im-
mediately after the addition of luciferase (40 mgmLÀ1 (final)) with stirring
at 258C. The slit width was 20 nm for emission and the photomultiplier
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Acknowledgements
This work was supported in part by research grants (grant nos. 19021010
and 19205021) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan to Y.U. H.T. was supported by a Grant-in-Aid for
JSPS Fellows. We thank T. Ozawa (Department of Chemistry, School of
Science, The University of Tokyo) for providing a plasmid encoding luci-
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Received: December 7, 2010
Published online: March 17, 2011
1810
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1800 – 1810