Synthesis of photoaffinity probe based on the leaf-opening factor from genus Albizzia

Article abstract of DOI:10.1016/j.tetlet.2008.04.002

The circadian rhythmic leaf-movements in legumes, called nyctinasty, are regulated by a pair of leaf-closing and -opening factors. Recent fluorescence studies revealed that cis-p-coumaroylagmatine (1), the leaf-opening factor from genus Albizzia, specific

Full text of DOI:10.1016/j.tetlet.2008.04.002

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3794–3796
Synthesis of photoaffinity probe based on the
leaf-opening factor from genus Albizzia
Masahiro Okada, Akira Matsubara, Minoru Ueda *
Tohoku University, Department of Chemistry, Aoba-ku, Sendai 980-8578, Japan
Received 29 January 2008; revised 31 March 2008; accepted 1 April 2008
Available online 8 April 2008
Abstract
The circadian rhythmic leaf-movements in legumes, called nyctinasty, are regulated by a pair of leaf-closing and -opening factors.
Recent fluorescence studies revealed that cis-p-coumaroylagmatine (1), the leaf-opening factor from genus Albizzia, specifically bound
to motor cells in a joint of leaf and stem. In order to identify the receptor protein, which is expected to be involved in the plasma
membrane of the cell, we designed and synthesized a photoaffinity probe (2), containing a photoreactive azide group on the aromatic
ring and FLAG segment as a peptide antigen for immunoprecipitation.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Albizzia saman; Nyctinasty; Leaf-opening factor; Photoaffinity probe
1. Introduction
affinity-labeling and affinity-purification with immunopre-
cipitation to be a useful approach for purifying the
receptor of a ligand. Specifically, immunoprecipitation
using the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-
Asp-Lys)7 is an effective method that is widely used for
the purification of a protein. Here, we report the synthesis
Most leguminous plants close their leaves in the evening,
as if sleeping, and open them in the morning. This circa-
dian rhythm, which is characterized by leaf-closing and
-opening movements and is known as nyctinasty, is regu-
lated by a pair of leaf-closing and -opening factors (LCF
and LOF, respectively).1,2 cis-p-Coumaroylagmatine (1)
was isolated as the LOF of the genus Albizzia.3 Recent
fluorescence studies revealed that 1 binds to motor cells
in the joint of the leaf and stem4 where they interacted with
specific receptors on the cells.5,6 These findings indicated
that 1 played a crucial role as an endogenous signaling
molecule in the control of nyctinastic leaf-movement
through binding to the corresponding receptor. However,
the receptor protein of 1 and the signaling pathway of
nyctinasty have not yet been resolved. In order to elucidate
the molecular mechanism responsible for nyctinastic leaf-
movement, identification of the receptor protein for 1 is
necessary. We considered the combination of photo-
OH
O
H
N
NH2
NH2
N
H
LOF 1
FLAG peptide
OH
(Gly)3 Asp-Tyr-Lys-(Asp)4-Lys OH
H
O
N3
O
N
NH2
N
H
NH2
LOF photoaffinity probe 2
*
Fig. 1. The chemical structure of LOF 1 and the LOF photoaffinity
probe 2.
Corresponding author. Tel./fax: +81 22 795 6553.
E-mail address: ueda@mail.tains.tohoku.ac.jp (M. Ueda).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.002

M. Okada et al. / Tetrahedron Letters 49 (2008) 3794–3796
3795
of photoaffinity probe 2, containing an azide on the aro-
matic ring as a photoreactive group and a FLAG segment
for use as an antigen for immunoprecipitation (Fig. 1).
UV irradiation for 5 min, the products were analyzed by
LC–MS (Fig. 3). Analog 3 was consumed completely,
and concurrently observed new signals at 254 nm were
weakened. This result indicated that the azide group in 3
was degradated by UV irradiation. Additionally, a new sig-
nal corresponding to a denitrogenated degradate of 3 was
mainly observed at m/z 290.0, and a minor signal, a water
adduct of the denitrogenated degradate of 3, was also
observed. Thus, 3 was confirmed to be degraded by UV
2. Results and discussion
We designed an efficient photoreactive probe in order to
identify the receptor for 1 using a photoaffinity-labeling
approach. In general, an aryl azide derivative such as azi-
dobenzoic acid is usually employed as a photoreactive
group.8 However, the addition of a large photoreactive unit
into a small molecule results in a marked increase in the
molecular size of the probe and would result in decreased
affinity between a ligand and the corresponding receptor.
Thus, we attempted to synthesize a novel photoaffinity
probe that contained a photoreactive group as a part of
the LOF molecule to minimize the increase in probe size.
First, we designed and synthesized the LOF analog (3),
which has an azide group on the aromatic ring of 1. The
molecular size of 3 is approximately the same as that of
1. The synthesis of 3 is summarized in Figure 2. The iodide
5 prepared from commercially available 4 in two steps was
coupled with propargyl alcohol by the Sonogashira reac-
tion9 to give 6 in an excellent yield. Reductions of 6 with
Na2S2O4 and then with Lindlar’s catalyst gave cis-olefin
(7). Successive treatment of 7 with triflyl azide gave 8.
The resulting 8 was oxidized to give 9 in 67% yield in
two steps.10 Finally, 9 was coupled with agmatine to give
3.11 We tested the biological activity of 3 using the leaves
of Albizzia saman according to a previous method.3 The
reason for using A. saman in bioassay is that A. saman will
be suitable for the future study on the identification of
receptor of LOF. A. saman would be most useful for the
collection of a large amount of motor cells among genus
Albizzia. Analog 3 was as effective as 1 in the bioassay
and showed leaf-opening activity at 300 lM.
50
0 min
UV = 254 nm
UV = 254 nm
A
25
0
50
5 min
0 min
5 min
1
B
C
D
25
0
100
m/z
318.1
50
0
100
m/z
black; 318.1
gray; 290.0
50
0
0
2
3
4
5
6
7
8
Time (min)
Fig. 3. Photodegradation analysis of the LOF analog
3
with UV
irradiation at 365 nm. (A) LC/ESI-MS analysis of the initial aqueous
solution containing the LOF analog 3 by UV at 254 nm and (C) the
extracted ion chromatogram m/z 318.1. (B) LC/ESI-MS analysis of
an aqueous solution containing LOF analog 3, which was irradiated for
5 min at 5.0 cm distance with UV light (365 nm and 1820 lW/cm2), by UV
at 254 nm and (D) the extracted ion chromatograms m/z 318.1
(represented by the black line) and m/z 290.0 (represented by the gray
line).
In order to confirm the photoreactivity of an azide in 3,
an aqueous solution containing 3 was irradiated at 5.0 cm
distance with UV light (365 nm and 1820 lW/cm2). After
N3Tf,
Et3N
1) Na2S2O4, H2O,
acetone, 58%
OPMB
OPMB
OH
OPMB
1) NaNO2, KI,
aq.HCl, 89%
2) PMBCl,
K2CO3,
DMF, 89%
CH2Cl2,
40%
2) H2, Pd/C-CaCO3,
quinoline, MeOH,
83%
H2N
N3
O2N
O2N
NH2
R
OH
OH
Propargyl alcohol,
Pd(PPh3)2Cl2,Et3N,
CuI, DMF, 98%
5: R = I
6: R =
4
7
8
OH
OPMB
1) Hydroxysuccimide,
DCC, CH2Cl2
OH
1) MnO2, CH2Cl2
H
2) NaClO2,
2-methyl-2-butene,
aq.NaHPO4,
2)
N
NH2
H2N
+
N3
N3
O
-
H
HSO4
NH2
CO2H
N
NH2
NH2
67% (in 2 steps)
N
H
acetone, aq.NaHCO3,
84% (in 2 steps)
9
LOF analog 3
3) TFA, CH2Cl2, 34%
Fig. 2. Synthesis of the LOF analog 3.

3796
M. Okada et al. / Tetrahedron Letters 49 (2008) 3794–3796
the photoreactive aryl azide unit in 2 is expected to interact
Lys(Boc)-
peptide synthesizer
with the binding site of the receptor. Thus, a higher yield
can be expected in cross-link formation by photo irradia-
tion. In addition, since several leaf movement factors pos-
sess a cis-p-coumaroyl moiety as well as 1,1,2 a similar
approach would be applicable to the detection of specific
receptors for other leaf-movement factors.
Arg(Pbf)-(Gly)3-FLAG(protection)-
10
1) 9, HBTU, HOBt, DIPEA,
NMP, rt
2) TFA/HFIP/CH2Cl2 (5/1/4)
0 ºC, dark, 4 h
References and notes
LOF photoaffinity probe 2
Fig. 4. Synthesis of the LOF photoaffinity probe 2.
1. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. 2000, 39, 1400.
2. Ueda, M.; Nakamura, Y.; Okada, M. Pure Appl. Chem. 2007, 79, 519.
3. Ueda, M.; Tashiro, C.; Yamamura, S. Tetrahedron Lett. 1997, 38,
3253.
4. Satter, R. L.; Gorton, H. L.; Vogelmann, T. C. The Pulvinus: Motor
Organ for Leaf Movement; American Society of Plant Physiologists,
1990.
5. Nagano, H.; Kato, E.; Yamamura, S.; Ueda, M. Org. Biomol. Chem.
2003, 1, 3186.
6. Nakamura, Y.; Matsubara, A.; Okada, M.; Kumagai, T.; Ueda, M.
Chem. Lett. 2006, 35, 744.
7. Hopp, T. P.; Prickett, K. S.; Price, V. L.; Libby, R. T.; March, C. J.;
Ceretti, D. P.; Urdal, D. L.; Conlon, P. J. Biotechnology 1988, 6, 1204.
8. Hatanaka, Y.; Sadakane, Y. Curr. Top. Med. Chem. 2002, 2, 271.
9. Sonogashira, K. In Metal-Catalyzed Cross-coupling Cross-Cou-
plingReactions; Diederich, F., Stang, P. J., Eds.; Wiley-VCH:
Weiheim, Germany, 1998; pp 203–229.
10. The intermediate 9: 1H NMR (500 MHz, CDCl3): d 7.66 (d, 1H,
J = 8.3 Hz), 7.30 (d, 2H, J = 8.6 Hz), 7.10 (d, 1H, J = 12.5 Hz), 6.92
(d, 2H, J = 8.6 Hz), 6.74–6.70 (m, 2H), 5.89 (d, 1H, J = 12.5 Hz), 5.01
(s, 2H), 3.81 (s, 3H); 13C NMR (125 MHz, CDCl3): d 171.3, 160.8,
159.6, 140.7, 140.1, 132.7, 129.3, 128.1, 119.0, 118.1, 114.1, 110.6,
104.6, 70.1, 55.3; ESI-HRMS calcd for C17H15N3O4Na [M+Na]+
348.0955, found 348.0954.
11. The LOF analog 3: 1H NMR (500 MHz, CD3OD): d 7.39 (d, 1H,
J = 8.5 Hz), 6.77 (d, 1H, J = 12.4 Hz), 6.61 (d, 1H, J = 2.3 Hz), 6.53
(dd, 1H, J = 8.5, 2.3 Hz), 5.88 (d, 1H, J = 12.4 Hz), 3.22–3.19 (m,
2H), 3.18–3.16 (m, 2H), 1.55–1.52 (m, 4H); 13C NMR (125 MHz,
CD3OD): d 170.0, 160.6, 158.6, 140.9, 133.6, 132.8, 123.3, 119.8,
113.0, 105.6, 42.1, 39.6, 27.5, 27.2; ESI-HRMS calcd for C14H19N7O2
[M+H]+ 318.1673, found 318.1672.
light immediately and was demonstrated to be a good
photoreactive analog of 1.
Based on the successful degradation of 3, we designed an
efficient probe for photoaffinity-labeling and immunopre-
cipitation of the receptor for 1. Although biotinylated
photoaffinity probes are usually used to identify binding
proteins of small bioactive molecules,8,12 it has been
reported that purification based on biotin–avidin chemistry
is often associated with high non-specific binding due the
prevalence of numerous endogenous biotinylated proteins
in the cell.12,13 We designed photoaffinity probe 2 using a
FLAG segment as an antigen because the FLAG peptide
is widely used as a peptide tag for immunoprecipitation
purification using anti-FLAG monoclonal antibody.7 Since
it is already known that the substitution of the agmatine
moiety in 1 with arginine does not affect biological activ-
ity,5,6 the FLAG peptide was introduced through peptide
linkage. The synthesis of 2 was easily achieved using
solid-phase peptide synthesis (Fig. 4). The protected
dodecapeptide (10) on the polymer support was coupled
with intermediate 9. After acidic cleavage from the resin,
the mixture was purified by HPLC to give the desired
photoaffinity probe (2).14 In a bioassay, probe 2 was
approximately half as effective as 1, and showed potent
leaf-opening activity in A. saman at 500 lM. Despite the
addition of the large FLAG segment, probe 2 retained its
leaf-opening activity, which is three-fifth strong as that of
natural LOF. This relative biological activity would be
enough for future study on the purification of receptor of
LOF.6 Thus, we successfully synthesized biologically active
photoaffinity probe 2 containing an aryl azide unit and a
FLAG segment.
12. Sugimoto, T.; Fujii, T.; Idutu, Y.; Yamamura, S.; Ueda, M.
Tetrahedron Lett. 2004, 45, 335.
13. Masuoka, J.; Guthrie, L. N.; Hazen, K. C. Microbiology 2002, 148,
1073.
14. The LOF photoaffinity probe 2: 1H NMR (500 MHz, D2O): d 7.16 (d,
1H, J = 8.1 Hz), 7.07 (d, 2H, J = 8.2 Hz), 6.90 (d, 1H, J = 12.5 Hz),
6.81 (d, 2H, J = 8.2 Hz), 6.73 (s, 1H), 6.62 (d, 1H, J = 8.1 Hz), 6.07
(d, 1H, J = 12.5 Hz), 4.62–4.54 (m, 5H), 4.49 (t, 1H, J = 6.8 Hz), 4.30
(dd, 1H, J = 7.4, 6.6 Hz), 4.20–4.15 (m, 2H), 4.03–3.92 (m, 6H), 3.05–
2.94 (m, 10H), 2.76–2.67 (m, 3H), 2.66–2.59 (m, 4H), 2.50 (dd, 1H,
J = 8.7, 15.2 Hz), 1.84–1.63 (m, 8H), 1.39–1.27 (m, 8H); ESI-HRMS
calcd for C62H88N20O26 [M+2H]2+ 764.3084, found 764.3086.
Given that a previous study revealed that the cis-p-cou-
maroyl moiety of 1 was essential for leaf-opening activity,5