Syntheses of novel photoaffinity probes for bioorganic studies on nyctinasty of leguminous plants

Article abstract of DOI:10.1016/S0040-4039(02)01460-0

Novel and non-radioactive photoaffinity probes (1 and 2) for the bioorganic study of nyctinasty are designed and synthesized based on potassium isolespedezate (3), which induce leaf-opening against the leaf of Cassia mimosoides L. These probes bear a trifluoromethyldiazirine or diazophenyl group for photoaffinity and a biotin subunit for affinity chromatography and chemiluminescent detection. Probes (1 and 2) showed leaf-opening activity at 5×10^-5 mol/L with leaves of C. mimosoides; thus, they would be an important tool for the identification of a receptor protein for 3.

Full text of DOI:10.1016/S0040-4039(02)01460-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6529–6532
Syntheses of novel photoaffinity probes for bioorganic studies on
nyctinasty of leguminous plants
Takanori Sugimoto,a Tomohiko Fujii,a Yasumaru Hatanaka,b Shosuke Yamamuraa and
Minoru Uedaa,*
aLaboratory of Natural Products, Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi,
Yokohama 223-8522, Japan
bResearch Institute for Wakan-yaku, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-01, Japan
Received 18 June 2002; accepted 17 July 2002
Abstract—Novel and non-radioactive photoaffinity probes (1 and 2) for the bioorganic study of nyctinasty are designed and
synthesized based on potassium isolespedezate (3), which induce leaf-opening against the leaf of Cassia mimosoides L. These
probes bear a trifluoromethyldiazirine or diazophenyl group for photoaffinity and a biotin subunit for affinity chromatography
and chemiluminescent detection. Probes (1 and 2) showed leaf-opening activity at 5×10−5 mol/L with leaves of C. mimosoides;
thus, they would be an important tool for the identification of a receptor protein for 3. © 2002 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. This circadian rhythm is known to be con-
trolled by the biological clock of such plants. We have
identified several bioactive substances that regulate this
leaf-movement, and our recent studies revealed the
mechanism for the control of nyctinasty by the biologi-
cal clock.1 The next issue is to explore how these
compounds induce leaf-movement.
The next issue of our research is focused on investiga-
tion of the role of leaf-movement factors in leaf-move-
ment, especially, the perception of the leaf-movement
factor by its receptor and the following signal transduc-
tion. Exploration of receptor molecule is the starting
point for a better understanding of how leaf-movement
factors function. Thus, it was strongly desired to
develop novel probe compounds for the identification
and characterization of receptor protein for the leaf-
movement factor. Photoaffinity labeling can provide
important information about the binding protein for
bioactive substances of low molecular weight.3 In the
photoaffinity labeling experiments, a trifluoromethyldi-
azirine or phenyl azide group is usually used as a
photoaffinity group.3 Now, we have synthesized two
photoaffinity probes (1 and 2) designed on the structure
of 3 for the detection and identification of a receptor
protein for 3.
A recent fluorescence study using fluorescence labeled
probe compound designed on the structure of potas-
sium isolespedezate (3), which induces leaf-opening
against the leaf of Cassia mimosoides L., revealed that
the target cell for 3 is motor cells.2 Motor cells, which
are contained in pulvini, are most important for leaf-
movement because their change in volume induces leaf-
movement. Leaf-opening substance 3 is supposed to
induce the swelling and shrinking of motor cells by
direct interaction with them to control leaf-movement.
Because of its hydrophobicity, 3 is supposed not to be
able to pass through the plasma membrane of a motor
cell. Thus, it was assumed that some receptors for 3
would exist on a plasma membrane of the motor cell of
C. mimosoides.
Both of our synthetic photoaffinity probes have a pho-
toaffinity group and a biotin unit in the molecule: one
has a trifluoromethyldiazirine group and the other has
a phenyl azide group for photoaffinity labeling, and
both of them have a biotin subunit for purification of
the binding protein by affinity chromatography and
chemiluminescent detection. These biotinyl pho-
toaffinity probes enable a non-radiochemical detection
of labeled protein on a polyvinylidene difluoride
(PVDF) membrane.4
* Corresponding author. Tel.: +81-45-566-1702; fax: +81-45-566-1697;
e-mail: ueda@chem.keio.ac.jp
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01460-0

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T. Sugimoto et al. / Tetrahedron Letters 43 (2002) 6529–6532
The most important issue is the introduction of large
photoaffinity and biotin groups with retention of bioac-
tivity. The molecular design of photoaffinity probes
followed that of previously reported fluorescence-
labeled probes. From the structure–activity relationship
study of 3, it was revealed that structure modification in
the sugar moiety of 3 did not diminish its bioactivity;
thus, we introduce these large functionalities into the
6-position of the sugar moiety. Also, we used galac-
toisolespedezate (4) instead of 3 to circumvent enzy-
matic hydrolysis of the glycosidic linkage in the plant
body.2
Synthesis of photoaffinity probe 1 was carried out
according to the synthetic route in Scheme 1. For the
sake of later oxidation, the primary hydroxy group of
galactose should be protected by a different protective
group from secondary hydroxy groups. Thus, 6-O-ben-
Scheme 1. Synthesis of photoaffinity probe 1.

T. Sugimoto et al. / Tetrahedron Letters 43 (2002) 6529–6532
6531
zyl diacetonegalactose (6) was synthesized by selective
acetonide formation between secondary hydroxy
groups in 5 using copper sulfate with catalytic sulfuric
acid5 and following protection of the residual primary
hydroxy group with a benzyl group. After acid treat-
ment and following acetylation, compound 7, which
has different protective groups on primary and sec-
ondary hydroxy groups, was obtained. Coupling of 8
with 9 gave 10, and then the product was subjected to
deacetylation, and the resulting secondary hydroxy
groups were protected with TBSOTf to give 11. After
conversion of the protective group on the 6-position of
the galactose moiety in 11 from the benzyl to an acetyl
group, resulting 12 was treated with excess amount of
DDQ (7 equiv.) for 2 days to give 13. This oxidation
gave an 8:1 mixture of (Z)- and (E)-13. The stereo-
chemistry was assigned by NOE correlation observed in
(Z)-13 (Scheme 1). These stereoisomers were separable
by column chromatography. The selective deacetylation
of 13 was achieved by carrying out the reaction at
−20°C. When the reaction was carried out at higher
temperature, the TBS group on the phenolic hydroxy
group was deprotected simultaneously. Then, the result-
ing primary hydroxy group was oxidized to give alde-
hyde (14). Coupling reaction of aldehyde (14) with 16
which has a trifluoromethydiazirine and biotin group4
was carried out in aqueous methanol, and the following
deprotections and purification by HPLC using
Develosil C8 UG-5 column gave the two isomers on the
oxime moiety of photoaffinity probe 1 (0.8 mg of (E)-1
and 0.8 mg of (Z)-1, respectively).6
Scheme 2. Synthesis of photoaffinity probe 2.
result suggests that no false binding to the protein
contained in neighboring cells was observed. This
highly selective binding will be also expected in our
photoaffinity probes that were synthesized according to
the same molecular design as fluorescent probes. Thus,
our bioactive photoaffinity probes would be highly
useful for the detection and identification of a receptor
protein for 3 on the motor cell. With a large quantity of
plasma membrane fraction collected by ultracentrifuga-
tion of plant extract, the exploration of a receptor
molecule for 3 will be carried out using these pho-
toaffinity probes.
Photoaffinity probe 2 was synthesized according to the
procedure reported in Ref. 2. Coupling of amine (17)2
with sulfosuccinimidyl [2-6-(biotinamido)-2-(p-azi-
dobenz-amido)-hexanoamido]ethyl-1,3%-dithiopropion-
ate (sulfo SBED)7 which has phenyl azide and biotin
groups gave 2.4 mg of 2 (Scheme 2).8
Acknowledgements
Both of the resulting photoaffinity probes 1 and 2
showed leaf-opening activity against the leaf of C.
mimosoides at 5×10−5 M (Table 1). (E)-1 was as effec-
tive as (Z)-1 on the bioassay. The bioactivity was about
one-tenth of that of natural product (3). Thus, we have
succeeded in the development of bioactive photoaffinity
probes for the bioorganic studies of nyctinasty.
The authors are indebted to 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, and Moritani Foundation for
financial support.
In previous fluorescence studies,2 our fluorescence-
labeled probes bound to motor cells selectively. This
Table 1. Time-course change of the status of the leaves treated with 5×10−5 M of 1, 2, and 3
Status of the leaves
16:00
17:00
18:00
19:00
20:00
21:00
22:00
1
2
3
++
++
++
++
++
++
++
++
++
++
++
+−
++
++
++
−−
++
++
++
−−
++
++
++
−−
++
++
++
−−
Blank
Status of the leaf was represented by the following marks: ++ completely open; + nearly open; +− at random; − nearly closed; −− completely
closed.

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T. Sugimoto et al. / Tetrahedron Letters 43 (2002) 6529–6532
CD3OD, rt): 7.76 (2H, d, J=8.8 Hz), 7.49 (1H, d, J=6.3
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d, J=13.7 Hz), 5.07 (1H, d, J=13.7 Hz), 4.59 (1H, br),
4.44 (1H, dd, J=4.4, 7.8 Hz), 4.25 (1H, dd, J=4.4, 7.4
Hz), 4.14–4.09 (3H, m), 3.85–3.78 (3H, m), 3.68–3.55 (5H,
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J=6.8 Hz), 1.74–1.50 (4H, m), 1.42–1.34 (2H, m) ppm.;
HR FAB MS (negative): [M−K]− Found m/z 925.2906,
C40H48O14F3N6S requires m/z 925.2901; IR (film) w: 3309,
1687, 1650, 1645, 1608, 1579 cm−1; [h]D22 +82.8 (c 0.53,
MeOH).
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6. (Z)-1: 1H NMR (400 MHz, CD3OD, rt): 7.76 (2H, d,
J=8.8 Hz), 7.33 (1H, d, J=7.8 Hz), 6.85 (1H, s), 6.82 (1H,
d, J=7.8 Hz), 6.74–6.67 (3H, m), 5.13 (2H, s), 4.65 (1H,
dd, J=1.4, 4.9 Hz), 4.44 (1H, dd, J=4.4, 7.3 Hz), 4.25
(1H, dd, J=4.4, 7.8 Hz), 4.17–4.10 (3H, m), 3.87–3.78
(3H, m), 3.70–3.54 (3H, m), 3.51 (2H, t, J=5.8 Hz), 3.15
(1H, m), 2.88 (1H, dd, J=4.9, 12.7 Hz), 2.66 (1H, d,
J=12.7 Hz), 2.18 (2H, t, J=7.3 Hz), 1.75–1.48 (4H, m),
1.44–1.31 (2H, m) ppm; HR FAB MS (negative): [M−K]−
Found m/z 925.2872, C40H48O14F3N6S requires m/z
1
8. 2: H NMR (400 MHz, CD3OD, RT): 7.95 (2H, d, J=8.8
Hz), 7.75 (2H, d, J=8.8 Hz), 7.15 (2H, d, J=8.8 Hz), 6.79
(1H, s), 6.73 (2H, d, J=8.8 Hz), 4.53–4.43 (2H, m), 4.26
(1H, dd, J=4.4, 7.8 Hz), 3.78 (3H, t, J=7.8 Hz), 3.73 (1H,
d, 3.4 Hz), 3.44–3.55 (6H, m), 2.90 (1H, dd, J=4.9, 12.7
Hz), 2.83–2.75 (4H, m), 2.67 (1H, d, J=12.7 Hz), 2.39
(2H, t, J=7.3 Hz), 2.17 (2H, t, J=7.8 Hz), 1.79–1.98 (2H,
m), 1.24–1.78 (10H, m) ppm; HR FAB MS (negative):
[M−K]− Found m/z 1002.3184, C43H56N9O13S3 requires
m/z 1002.3160; IR (film) w: 3296, 2126, 1644, 1604 cm−1
[h]2D3 +68.4 (c 0.80, MeOH).
;
925.2901; IR (film) w: 3309, 1685, 1650, 1608, 1578 cm−1
[h]2D2 +66.1 (c 0.62, MeOH); (E)-1: 1H NMR (400 MHz,
;