Synthesis of a novel bioactive photoaffinity probe based on a leaf-movement factor with potential high binding affinity to its receptor molecule

Article abstract of DOI:10.1016/S0040-4039(03)00287-9

Nyctinastic leaf-movement is induced by the binding of the leaf-movement factor with a motor cell located in the pulvini of the plant. Some receptors of the leaf-movement factor should be involved in this biological event. We developed a novel photoaffini

Full text of DOI:10.1016/S0040-4039(03)00287-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2497–2499
Synthesis of a novel bioactive photoaffinity probe based on a
leaf-movement factor with potential high binding affinity
to its receptor molecule
Tomohiko Fujii, Takanori Sugimoto, Shosuke Yamamura and Minoru Ueda*
Laboratory of Natural Products, Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi,
Yokohama 223-8522, Japan
Received 15 January 2003; revised 27 January 2003; accepted 31 January 2003
Abstract—Nyctinastic leaf-movement is induced by the binding of the leaf-movement factor with a motor cell located in the
pulvini of the plant. Some receptors of the leaf-movement factor should be involved in this biological event. We developed a novel
photoaffinity probe (1) for the detection of receptor molecules. Probe compound 1 bears a photolabeling group on the 2%-position
of the glycon moiety, which is near the potential binding site of the molecule with its receptor. This molecular design would be
effective for the improvement of photolabeling yield. © 2003 Elsevier Science Ltd. All rights reserved.
Most leguminous plants close their leaves in the
evening, as if to sleep, and open them early in the
morning according to the circadian rhythm controlled
by the biological clock. Extensive studies on nyctinastic
plants led to the isolation of a variety of leaf-closing
and leaf-opening substances. We found that the biolog-
ical clock regulates the balance of concentration
between leaf-opening and -closing substances in the
plant body during the day.1 We also revealed that some
receptors for this leaf-movement factor are located on
motor cells,2–4 which plays a key role in nyctinastic
leaf-movement.5 Recently, we succeeded in synthesizing
biologically active photoaffinity probe (2) to look for
the native factor receptor based on potassium les-
pedezate (3),6 which is a leaf-opening substance of
Cassia mimosoides.7 However, probe 2 was designed to
bear a photoaffinity group on the 6%-position of the
sugar moiety: thus the aglycon moiety, which is impor-
tant for bioactivity and expected to be a binding site
with its receptor, is far from the photoaffinity group
(Fig. 1).
In this paper, we report a novel photoaffinity probe (1)
which bears a photolabeling group on the 2%-position of
the sugar moiety and is expected to have high photola-
beling yield in the binding examination with its recep-
Figure 1. Molecular design of the photoaffinity probe (1).
* Corresponding author. Tel.: +81-45-566-1702; fax: +81-45-566-1697; e-mail: ueda@chem.keio.ac.jp
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00287-9

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T. Fujii et al. / Tetrahedron Letters 44 (2003) 2497–2499
Scheme 1. Synthetic route of photoaffinity probe (1).
tor. Probe 1 was designed according to the molecular
design of 2. However, we used galactosamine which has
an amino group on the 2%-position to introduce the
photolabeling unit. Amide linkage was used to connect
the photolabeling unit with the glycon part. This molec-
ular design would be effective for the improvement of
labeling yield because the photolabeling group on the
2%-position is extruded to the direction of aglycon which
is expected to be a binding site with the receptor. This
change in molecular design requires revision of the
synthetic route that was used in 2.
unit bearing trifluoromethydiazirine and biotin, which
is separately prepared according to the method by
Hatanaka.9 Free amine, which was obtained in the
deprotection of 11, was used in the coupling reaction
without purification. t-Butyl ester in 11 was essential
for the synthesis of 12. When we used the methyl ester
(13) instead of 11, we obtained lactam (14)10 [lH-2% 3.63
ppm] quantitatively in the following deprotection of the
amino group on the 2%-position (Scheme 2). The t-butyl
group would prevent the formation of lactam by its
steric hindrance.
However, deprotection of the t-butyl ester in 12 raises a
serious problem. We examined the reaction conditions
on the deprotection of 12 thoroughly. But the use of
several weak acids,11 such as formic acid, acetic acid,
TFA, and benzoic acid gave a complex mixture of
decomposed products or ended in no reaction. The
resulting complex mixture mainly contained the frag-
The synthetic route of probe 1 is summarized in
Scheme 1. According to the method by Lee,8 we syn-
thesized 3,4,6-tri-O-acetyl-2-deoxy-2-phtalimido-a,b-D-
galacto-pyranosyl bromide (4). Compound 4 was cou-
pled with 7 by using AgOTf. After conversion of the
benzyl group in resulting 8 to a TBS group, DDQ
oxidation was carried out to give 10. Compound 10 was
treated with TBAF, and then sodium methoxide to give
11. Compound 11 was deprotected with hydrazine
monohydrate, and then coupled with the photolabeling
Scheme 2. The formation of lactam in the deprotection of
phtalimido group.

T. Fujii et al. / Tetrahedron Letters 44 (2003) 2497–2499
2499
ments obtained by the dissociation of the glycosidic
Acknowledgements
bond or the amido bond. After many trials, we found
that the treatment of 12 by neat TFA within 30 s at rt
provided good results. The main product was a free
carboxylate form of 1 (82%) with a small amount of the
recovered starting material (9%). After subsequent
treatment with sodium carbonate, photoaffinity probe
112 was obtained.
This work was supported by the Ministry of Education,
Science, Sports and Culture (Japan) for Grant-in-Aid
for Scientific Research (No. 1302467 and 12680598),
Pioneering Research Project in Biotechnology given by
the Ministry of Agriculture, Forestry and Fisheries,
Kato Memorial Bioscience Foundation, Naito Founda-
tion, Kurata Foundation, Moritani Foundation. This
work was also supported by Grant-in-Aid for the 21st
Century COE program ‘KEIO Life Conjugate Chem-
istry’ from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
Probe 1 was effective for the leaf-opening of C. mimo-
soides at 8×10−5 M. Thus, the bioactivity of 1 was
one-eightieth as strong as that of the native factor. The
decrease in bioactivity compared with probe 3 could be
due to the steric hindrance in the binding with the
receptor molecule (Fig. 1).
References
Photodegradation of probe 1 was also examined. UV
light (u 365 nm) was irradiated to an aqueous solution
of 0.33 mM 1 for 60 min. The reaction was monitored
by the decrease in the peak intensity at 360 nm in UV
spectrum of the reaction mixture, that corresponds to
the absorption of the trifluoromethyldiazirine group.
The reaction mixture was then dried up and analyzed
by using negative-mode FAB MS to give the peak at
m/z 915 which corresponds to [15-H]− ion. This result
showed that probe 1 could react with the receptor
molecule by the radiation of UV light for 15 min.
1. Ueda, M.; Takada, N.; Yamamura, S. Bull. Chem. Soc.
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2. Lee, Y. In The Pulvinus in Motor Organ for Leaf Move-
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3. Ueda, M.; Wada, Y.; Yamamura, S. Tetrahedron Lett.
2001, 42, 3869–3872.
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mura, S. Tetrahedron 1990, 46, 383–394.
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Res. 1987, 170, 27–46.
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Med. Chem. 1994, 12, 1367–1373.
1
10. 14 H NMR (400 MHz, CD3OD, rt): 7.64 (2H, d, J=8.8
Hz), 6.74 (2H, d, J=8.8 Hz), 6.71 (1H, s), 4.86 (1H, d,
J=7.1 Hz), 3.91 (1H, dd, J=8.3, 12.5 Hz), 3.87 (1H, dd,
J=0.7, 2.7 Hz), 3.81 (1H, dd, J=4.6, 12.5 Hz), 3.80 (1H,
dd, J=0.7, 4.6 Hz), 3.66 (1H, dd, J=2.9, 10.7 Hz), 3.63
(1H, dd, J=7.1, 10.7 Hz) ppm.; FAB MS (positive) m/z
The molecular design of photolabeling probes involves
an ambivalent issue: the nearer the large photolabeling
group is placed to the potential binding site of the
probe molecule, the weaker the bioactivity of the pho-
toaffinity probe becomes. In past photolabeling studies,
molecular design of the probe requires the cost of either
high binding affinity or biological activity because these
two factors are never compatible. This is the most
important problem in the photolabeling studies using
probe compound. However no example has been
reported to deal with this issue to evaluate the effectives
of these two factors.
324 [M+H]+: IR (film) w: 1670, 1606, 1512, 1441 cm−1
.
11. Chandrasekaran, S.; Kluge, A. F.; Edwards, J. A. J. Org.
Chem. 1977, 42, 3972–3974.
1
12. 1: H NMR (270 MHz, CD3OD, rt): 7.88 (1H, d, J=8.2
Hz), 7.65 (2H, d, J=8.6 Hz), 6.96 (1H, d, J=8.2 Hz),
6.80 (2H, s), 6.66 (2H, d, J=8.6 Hz), 5.32 (1H, d, J=8.6
Hz), 4.43 (2H, m), 4.25 (3H, m), 3.92–3.78 (5H, m),
3.73–3.45 (11H, m), 2.90 (1H, dd, J=4.9, 12.9 Hz), 2.67
(1H, d, J=12.9 Hz), 2.18 (2H, t, J=7.5 Hz), 1.66–1.41
(6H, m) ppm; 13C NMR (100 MHz, CDCl3, rt): 172.3,
165.8, 165.8, 162.8, 157.2, 156.5, 131.6, 131.4, 131.0,
128.4, 125.9, 125.4, 123.1, 120.4, 118.9, 115.0, 111.3, 99.5,
77.6, 75.8, 75.5, 73.2, 69.8, 69.6, 69.2, 68.6, 67.2, 61.1,
60.1, 59.2, 55.4, 53.8, 38.4, 35.1, 29.0, 28.2, 28.0, 25.3
ppm.; HR-FAB MS (negative): [M−K]−. Found m/z
925.2899, C40H48O14N6F3S requires m/z 925.2901; IR
(film) w: 1693 1651, 1608, 1549, 1512 cm−1; [h]D24 −21.2° (c
0.5, MeOH).
We resolved this issue by using two photoaffinity
probes designed on different concepts. Now, we synthe-
sized two types of photoaffinity probes designed on
different concepts: probe 1 was designed for high label-
ing yield with the receptor molecule, whereas probe 2
was designed for high affinity with the receptor by
reducing the steric hindrance by large photolabeling
group. The detection of receptor molecules using these
two probes is now in progress.