Organic Letters
Letter
demethylated product 21 quantitatively. Unexpectedly, trans-
formation of amide 21 into v-CTM (6) by deacetylation was
efficiently accomplished under mild conditions, such as
treatment with sodium bicarbonate in refluxing methanol,
which could be due to assistance from the phenolic hydroxy
group through extended π-conjugation. Together, the synthesis
of v-CTM (6) was achieved in 9 steps from commercially
available pyrazine 9 with a 15% overall yield.
Table 1. Luminescence Properties of CTZ Analogs as a
Substrate of RLase and RLase-547
RLase
RLase-547
λmax
b
b
λmax
a
c
a
c
Imax [Int.]
[FWHM]
(nm)
Imax [Int.]
[FWHM]
(nm)
CTZ analog
(%)
(%)
d
e
CTZ (1)
100
485
100
547
f
g
[100]
[95]
519
[100]
[124]
593
v-CTZ (5) and a C2-modified analog were successfully
synthesized by the cyclocondensation reaction of v-CTM (6)
with α-ketoacetals (Scheme 6). Although v-CTZ (5) is highly
v-CTZ (5)
71.8
213
[47.3]
18.9
[105]
526
[73.4]
16.9
[130]
599
cf3-v-CTZ
(24)
[12.3]
[121]
[11.9]
[132]
Scheme 6. Synthesis of v-Coelenterazines
a
The maximum intensity of luminescence (Imax) and the integrated
value of luminescence (Int.) are obtained in 0.1 s intervals for 60 s, and
the relative activity is expressed as a percentage of the mean value with
b
respect to coelenterazine (n = 4). All bioluminescence spectra are
c
corrected according to the manufacture’s protocol. FWHM = full
d
e
f
width at half-maximum. 9.4 × 107 rlu/μg. 6.9 × 106 rlu/μg. 2.9 ×
g
1010 rlu/μg. 3.7 × 109 rlu/μg (rlu = relative light units).
of the coupling partners. Further studies on CTZ analogs and
CTZ-utilizing luciferases will allow the development of efficient
bioimaging systems, including in vivo imaging of living animals.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization data including
copies of NMR spectra. The Supporting Information is
susceptible to oxidation and unstable in solutions, heating a
mixture of v-CTM (6) with α-ketoacetal 22 in acidic aqueous
ethanol provided v-CTZ (2) in 13% yield. The spectral data for
chromatographically purified v-CTZ (5) were identical to those
reported in the literature.4a We previously reported that some
C2-modified CTZ analogs, such as cf3-CTZ (2), gained
significantly improved stability in a buffer solution.7c Based on
this observation, we also prepared a trifluoromethyl analog of v-
CTZ, where cyclocondensation of v-CTM (6) with α-
ketoacetal 237b afforded the desired cf 3-v-CTZ (24) in 29%
yield. Although further studies to improve the efficiency of the
cyclocondensation step are needed, the isolated yields indicated
that the stability of cf 3-v-CTZ (24) compared to v-CTZ (5)
was improved, exhibiting a similar trend with our previous
report for C2-modified CTZ analogs.7b,c
AUTHOR INFORMATION
■
Corresponding Author
Present Address
⊥Chemical Biology Team, Imaging Chemistry Group, Division
of Bio-Function Dynamics Imaging, RIKEN Center for Life
Science Technologies (CLST), 6-7-3 Minatojima-minamima-
chi, Chuo-ku, Kobe 650-0047, Japan.
Notes
The authors declare no competing financial interest.
The newly synthesized cf3-v-CTZ (24) was a good substrate
for RLase (Table 1). The luminescence properties of the
synthesized v-CTZ (5) were in good agreement with the results
of previous reports,5,9 where remarkable red-shifted emission
spectra were obtained when it was used as a substrate for RLase
(λmax = 485 nm shifted to 519 nm) or RLase-547 (λmax = 547
nm shifted to 593 nm) compared with native CTZ (1). Slightly
increased red shifts, λmax = 526 and 599 nm for RLase and
RLase-547, respectively, were observed for cf 3-v-CTZ (24).
The decreased luminescence intensities were observed for cf 3-
v-CTZ (24) compared with v-CTZ (5). We previously
observed a similar trend between cf3-CTZ (2) and CTZ
(1),7c suggesting that the modification of the 4-hydroxybenzyl
group at the C2 position of CTZs to 4-(trifluoromethyl)benzyl
group affects the luminescence properties.
ACKNOWLEDGMENTS
■
The authors thank Ms. Ayako Hosoya at Tokyo Medical and
Dental University for HRMS analyses. This work was
supported by Platform for Drug Discovery, Informatics, and
Structural Life Science from MEXT and AMED, Japan, JSPS
KAKENHI Grant Numbers 26560443 (T.H.) and 26350971
(S.Y.), and grants from The Noguchi Institute, Takeda Science
Foundation, The Uehara Memorial Foundation (S.Y.), The
Kurata Memorial Hitachi Science and Technology Foundation,
and The Naito Foundation (T.H.).
REFERENCES
■
(1) (a) Shimomura, O. Bioluminescence: Chemical Principles and
Methods; World Scientific Publishing: Singapore, 2006; pp 159−179.
(b) Teranishi, K. Bioorg. Chem. 2007, 35, 82−111.
(2) (a) Tsuji, F. I.; Ohmiya, Y.; Fagan, T. F.; Toh, H.; Inouye, S.
In summary, we have developed a concise synthetic method
for v-CTZ using three sequential cross-couplings and RCM as
key reactions. The convergent approach should enable the
preparation of an array of v-CTZs through the simple exchange
C
Org. Lett. XXXX, XXX, XXX−XXX