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T. Sugimoto et al. / Tetrahedron Letters 45 (2004) 335–338
the receptor by reducing the steric hindrance by a large
photolabeling group. We examined the difficulty in the
photoaffinity labeling by using two photoaffinity probes
designed on different concepts. In this paper, we report
the detection of a receptor molecule for the leaf-move-
ment factor using these three probes, and advance a
guideline for the molecular design of probe compounds
for successful photoaffinity labeling.
Thus, we synthesized diamine from 12 as intermediate,
and then, biotin coupled with a triglycine linker was
0
introduced on the 6 position of the glycon moiety using
DCC, and then 4-bromomethylbenzophenone was
0
introduced on its 2 position. Removal of tert-butyl group
by using neat TFA gave probe 1. Probe 1 was effective
À4
for the leaf opening of C. mimosoides at 1 · 10 M that
is 100 times as weaker as the native factor (4). Probes 2
À5
À5
(
bioactivity: 8 · 10 M) and 3 (bioactivity: 5 · 10 M)
were also synthesized according to Ref. 9.
Photolabeling examination needed a large amount of
motor cells containing the receptor because of its
extremely low content. We collected a large amount of
plant motor cells by cutting a large number of sections
of plant pulvini (size: ca. 1 mm · 1 mm) containing the
motor cell one by one under a stereoscopic microscope.
One cross-linking experiment needed about 1000 plant
sections. Successive homogenization in extraction buffer
(0.25 M sucrose, 3 mM EDTA, 2.5 mM DTT, 25 mM
Tris–MES, pH 7.8) at 4 ꢁC, filtration with nylon mesh,
and twice ultracentrifugation (1st: 3000 · g, 15 min, 4 ꢁC,
2
nd: 100,000 · g, 60min, 4 ꢁC) gave a pellet of the crude
membrane fraction. The content of protein in that
fraction was determined to be 134 lg by the Bradford
method with BSA as a reference. The membrane AT-
Pase activity, which is a reference for the purity of the
plasma membrane was determined to be 0.29 lmol/
10
mg min by SandstomÕs method. The crude membrane
fractions were suspended and incubated with 3 lM
aqueous solution of probes 1–3 for 20min at rt,
respectively. After cross-linking by irradiation of UV-
light (365 nm) for 6 min, the suspended membrane
fraction was solubilized by the addition of an electro-
phoresis buffer containing SDS. The membrane fraction
was analyzed by SDS–PAGE (7.5%T). After Western
blotting, detection of the bands of the potential receptor
for 4 was carried out by chemiluminescence detection
with ECL Advance Western Blotting Detection Kit
8
Synthesis of novel probe 1 was carried out according to
the route in Scheme 1. In this probe, a large benzo-
phenone group and a large biotin unit were separately
0
0
introduced on the 2 - and 6 -position of the sugar moi-
ety, respectively. tert-Butyl ester in 10 was essential for
the synthesis of 14. In the synthesis of probe 2, which
was reported in Ref. 9, we experienced that the use of
methyl ester instead of 10, lactam was obtained quan-
titatively in the following deprotection of the amino
group on the 2 -position. The tert-Butyl group would
prevent the formation of lactam by its steric hindrance.
9
(Amersham Bioscience Co. Ltd.), which is a method for
the detection of bands of biotinylated proteins. Photo-
labeling experiments with probes 1 and 2 gave two bands
corresponding to binding proteins for 4. One is due to a
protein of 210kDa molecular weight, and the other to a
protein of 180kDa (lane 2 in Fig. 1). The molecular
weight was estimated from comparison with a biotinyl-
ated molecular weight marker (Amersham Bioscience
Co. Ltd.).
0
Specific bindings of the probes were confirmed by the
disappearance of the corresponding bands in the cross-
linking in the presence of 1000-fold molar excess of
nonlabeled leaf-opening substance 4 (lane 3 in Fig. 1).
The bands for proteins smaller than 100 kDa that were
observed in both lanes 2 and 3 in Figure 1 were concluded
to be nonspecific bands due to the biotin unit, because
they were also detected in the cross-linking examination
using photolabeling unit 5, which was not connected
with 4. The reproducibility was checked by labeling
experiments repeated 10times. On the other hand, no
specific band was detected in the labeling experiments
with probe 3 (Fig. 2). These results suggested that the
close arrangement of the photolabeling group and the
Scheme 1. Synthesis of probe 1.