54
M. Nakamura et al. / Tetrahedron Letters 46 (2005) 53–56
ester 28 as a mixture of geometrical isomers (E/Z = 3:1).
This mixture was separated by silica gel column chroma-
tography and the stereochemistry of each geometrical
isomer was determined by NOE experiments. The reduc-
tion of E-28 with DIBAH followed by the oxidation of
the resulting allylic alcohol 32 with MnO2 gave the alde-
hyde 36. The Baeyer–Villiger oxidation of 36 was
achieved using 60% hydrogen peroxide in t-amyl alcohol
in the presence of selenium dioxide to give the Latia
luciferin analogue 12. Analogues 13–15 were synthesized
from 2-bromo-m-xylene, 5-bromo-1,2,4-trimethylbenz-
ene, or 2-bromomesitylene in a manner similar to that
described.
O
H
O
CH3
O
O
Latia luciferin
1
3
X
O
H
O
O
O
X = H, NO2, CN, NMe2, OMe
9
4
5
6
7
8
O
O
O
O
H
H
The bioluminescence activity of 12–15 was measured by
a previously described procedure.4b Thus, frozen Latia
was homogenized in 50mM 2-amino-2-hydroxymethyl-
1,3-propanediol Tris–HCl buffer (pH7.6) at 0°C and
the homogenized mixture was centrifuged at 7000 rpm
for 20min at 4°C. The supernatant was diluted 10 times,
and used for the bioluminescence measurement as a
crude luciferase. The light production was measured
by mixing 100lL of the crude luciferase solution with
100lL of Latia luciferin or the analogue (60lM) in
25% ethanol solution.4b The photons were counted by
a luminometer luminescencer-PSN AB-2200 (Atto,
Tokyo, Japan) for 30min. The wavelength of luciferin
and its analogue was measured by a LumiFLSpectro-
Capture AB-1850 (Atto). The inhibitory activity was
measured by competition assay with natural luciferin.
The inhibitory activity was measured by mixing 100lL
of the crude luciferase solution with 100lL of 60lM
Latia luciferin and the analogue (various concentra-
tions) in 25% ethanol solution. The photons were
counted by a luminometer luminescencer-JNR II AB-
2300 (Atto) for 30min.
CH3
O
O
11
10
H
O
12
13
O
H
O
H
O
O
14
15
Scheme 2. The structures of Latia luciferin and its analogues.
similar to the vision pigment retinal, and the enol for-
mate functionality. Upon saturation of the cyclohexene
ring into the corresponding cyclohexane (analogue 9), or
upon replacement of the ring system by the phenyl
group (analogue 11), the bioluminescence activity disap-
peared, implying that the 2,6,6-trimethylcyclohexene
ring moiety is important for the substrate recognition
with the luciferase. While the corresponding enol ether
analogue (10) had no bioluminescence activity, the cor-
responding enol acetate 3 and enol benzoate analogues
4–8 exhibited substantial light production.4 In addition,
light production was delayed when the enol acetate and
enol benzoate analogues (3–8) were used.
The bioluminescence activity of the synthesized ana-
logues are summarized in Table 1. The analogues
E-13, E-14 and E-15 showed luminescent activity,
although these analogues were less potent than the
authentic4a Latia luciferin (1). The bioluminescence
activity of E-12 exhibited no significant differences in
the blank luminescent measurement similar to E-11,4a
while, upon increasing the substrate and luciferase, E-
12 exhibited a small luminescent activity (data not
shown). The bioluminescence spectra of E-13, E-14
and E-15 were identical with those of the authentic Latia
luciferin (1) (kmax 536nm) (Table 1), indicating that the
2,6,6-trimethylcyclohexene moiety of the luciferin does
not affect the bioluminescence spectra. The inhibitory
activity of the inactive analogues E-11 and E-12 against
the bioluminescence activity of the authentic Latia lucif-
erin (1) was measured (Table 2). The E-12 was found to
exhibit the same inhibitory activity as oxyluciferin, while
E-11 exhibited approximately 10 times less potency. It is
indicated that the recognition of E-11 and oxyluciferin
(2) for luciferase was the same, and supported the result
that E-11 had slightly bioluminescence and E-10 did not
have bioluminescence. These results indicated that the
number and the position of the methyl group(s) on the
phenyl ring are important for the substrate recognition
in the Latia luciferase. In addition, the presence of the
Herein we report the bioluminescence activity of the
Latia luciferin analogues 12–15 having methyl-substi-
tuted phenyl groups instead of the natural 2,6,6-trimeth-
ylcyclohexane ring system.
The luciferin analogues 12–15 were prepared as follows
(Scheme 3). The Heck reaction of 2-bromotoluene with
methyl vinyl ketone in the presence of Pd(OAc)2, PPh3
and Et3N, gave the a,b-unsaturated ketone 16, which
upon treatment with Pd black under a H2 atmosphere,
yielded a mixture of ketone 21 and alcohol 22. Oxida-
tion of the mixture of 21 and 22 with tetrapropylammo-
nium perruthenate (TPAP) furnished the pure ketone 21
in 82% yield from 16. The ketone 21 was subjected to the
Horner–Emmons reaction to afford the a,b-unsaturated
Nakamura, Mitsuhiro
