Azido-coronatine: A useful platform for "click chemistry"- mediated probe synthesis for bioorganic studies

Article abstract of DOI:10.1271/bbb.100518

We report on the development of azide-coronatine as a useful platform for azide alkyne cycloaddition ("click chemistry")-mediated synthesis of molecular probes. (p)-Azido-coronatine was synthesized in 10 steps with 11% yield using improved synthesis of coronafacic acid, in which the highly exo-selective Diels-Alder reaction (endo:exo > 1:25) is the key step. Azido coronatine was as effective as the original coronatine in a stomatal opening assay, and was easily modified to a fluorescein isothiocyanate (FITC)-labeled probe with high yield.

Full text of DOI:10.1271/bbb.100518

Biosci. Biotechnol. Biochem., 74 (10), 2092–2095, 2010
Azido-Coronatine: A Useful Platform for ‘‘Click Chemistry’’-Mediated
Probe Synthesis for Bioorganic Studies
y
Masahiro OKADA, Syuusuke EGOSHI, and Minoru UEDA
Department of Chemistry, Tohoku University, 6-3 Aramakiaza-Aoba, Aoba-ku, Sendai 980-8578, Japan
Received July 20, 2010; Accepted July 20, 2010; Online Publication, October 7, 2010
[doi:10.1271/bbb.100518]
We report on the development of azide-coronatine as
a useful platform for azide alkyne cycloaddition (‘‘click
chemistry’’)-mediated synthesis of molecular probes.
(þ)-Azido-coronatine was synthesized in 10 steps with
11% yield using improved synthesis of coronafacic acid,
in which the highly exo-selective Diels-Alder reaction
(endo:exo > 1:25) is the key step. Azido coronatine was
as effective as the original coronatine in a stomatal
opening assay, and was easily modified to a fluorescein
isothiocyanate (FITC)-labeled probe with high yield.
2 is required. Here, we report on the development of
azide-COR (5) as a useful platform for azide alkyne
cycloaddition (click chemistry11,12))-mediated synthesis
of useful derivatives, including molecular probes
(Fig. 1).
Recently, copper-catalyzed azide-alkyne cycloaddi-
tion (CuAAC)11,12) has been applied in the synthesis of
various functional molecules, including molecular
probes. The azide group is the most valuable bioorthog-
onal functionality.12) It is used for labeling certain target
proteins, which can be tagged subsequently by CuAAC
or copper-free click chemistry (CFCC)13–15) with fluo-
rescence-dye or biotin. In a previous paper,9) we
reported the synthesis of four stereoisomers of COR
using exo-selective Diels-Alder reaction at a key step.
We examined structure-activity relationship of COR in
stomatal opening activity, and reached the conclusion
that the coronafacic acid moiety is more important for
activity. Here the azide handle should be introduced into
the ethyl group of coronamic acid because of the
difficulty of chemical modification of carboxylate.
Key words: coronatine; click chemistry; molecular
probe; fluorescence; stomatal opening
Jasmonic acid (JA, 1) is an important plant hormone
involved in plant defensive behavior against biotic and
abiotic stresses, including pathogen attack.1) The mode
of action of jasmonate has been verified through
identification of the COI1-JAZ signaling module,2,3) in
which COI1 functions directly as JA receptor.4) Coro-
natine (COR, 2),5) a phytotoxin isolated from Pseudo-
monas syringae pv. atropurpurea, is a conjugate of
polyketide coronafacic acid and nonproteinogenic amino
acid coronamic acid. COR (2) is considered a mimic of
N-(ꢀ)-7-iso-jasmonoyl L-isoleucine (7-iso-JA-Ile, 3), a
genuine bioactive form of JA,6) and has played an
important part in the recent progress of JA research, as
in the discovery of the coi1 mutant7,8) and the identi-
fication of the JA receptor.2–4) However, it has remained
unclear whether COI1 is the exclusive receptor of 1
in its various bioactivities. For example, COR (2)
has stomatal opening activity9) for Ipomoea tricolor,
Commelina communis, and Arabidopsis thaliana, for
which it is unclear whether COI1 acts as a receptor. In
this research, the development of chemically modified
forms of 2, including fluorescence-labeled and photo-
affinity probes, is indispensable. Observing the structure
of 2, it is easily recognized that the most feasible
modification is one on the carboxylate functionality,
such as esterification or amide formation. However,
Toshima et al. reported that such modifications were
very difficult because condensation by using carbodii-
mide reagent resulted in intramolecular cyclization to
give anhydrocoronatine (4) with the oxazole ring
(Fig. 1).10) They concluded that modification of the
carboxylate of 2 is not promising for the attachment of a
linker. Hence, a practical method for the modification of
Materials and Methods
General procedure. The NMR spectra of the synthetic compounds
were recorded on a Jeol JNM-Lambda400 (1H (400 MHz) and 13C
(100 MHz), Jeol, Tokyo) using TMS as internal standard. The HRMS
spectra of DART-TOF were recorded on a DART (Jeol). The IR
spectra were recorded on a Jasco FT/IR-410 (Jasco, Tokyo). Specific
rotations were measured on a Jasco DIP-360 polarimeter (Jasco). Silica
gel column chromatography was performed on a Silica Gel 60N (Kanto
Chemical, Japan). HPLC purification and chiral analysis were
performed on a Jasco LC-2075 series HPLC system (Jasco). Reagents
and solvents were purchased from Kanto Chemical and Wako Pure
Chemical Industries.
(þ)-Azido-coronatine (5) and (ꢀ)-ent-azido-coronatine (ent-5). To
a
solution of (ꢀ)-14 (8.50 mg, 32.7 mmol), (ꢁ)-13 (6.80 mg,
32.7 mmol), and Et3N (6.61 mg, 65.3 mmol) in N-methylpyrrolidone
(NMP, 1.0 ml) was added O-(7-azabenzotrazol-1-yl)-1,1,3,3-tetrame-
thyluronium-hexafluorophosphate (HATU, 14.9 mg, 39.2 mmol) at 0 ꢂC
under an argon atmosphere. After stirring for 2 h, the reaction mixture
was quenched with 5% aqueous KHSO4. The mixture was extracted
with EtOAc (3 ꢃ 5 ml), and the resulting organic layer was washed
with saturated aqueous NaCl, dried over Na2SO4, and filtered. After
evaporation, the residue was purified by silica gel column chromatog-
raphy (n-hexane/EtOAc = 7/1–6/1) to give a mixture of condensa-
tion products. These compounds were dissolved in THF (0.16 ml) and
CH3OH (0.16 ml). To this solution was added 1 M aqueous LiOH
(0.16 ml). After stirreing for 2 h, the reaction mixture was quenched
y
To whom correspondence should be addressed. Fax: +81-22-795-6553; E-mail: ueda@m.tohoku.ac.jp
Abbreviations: JA, jasmonic acid; COR, coronatine; 7-iso-JA-Ile, N-(ꢀ)-7-iso-jasmonoyl L-isoleucine; CuAAC, copper-catalyzed azide-alkyne
cycloaddition; CFCC, copper-free click chemistry; FITC, fluorescein isothiocyanate; NMP, N-methylpyrrolidone; HATU, O-(7-azabenzotrazol-1-yl)-
1,1,3,3-tetramethyluronium-hexafluorophosphate

Synthesis of Azido-Coronatine
2093
Fig. 1. Structures of JA (1), (þ)-Coronatine (2), and (þ)-7-iso-JA-Ile (3), and Coupling Reactions with Molecular Tags: (A) Toshima et al. in 2004
Using 2, (B) This Study Using (þ)-Azido-Coronatine (5).
23
with 5% aqueous KHSO4. This mixture was extracted with EtOAc
(3 ꢃ 5 ml), and the resulting organic layer was washed with saturated
aqueous NaCl, dried over Na2SO4, and filtered. After evaporation, the
residue was purified by silica gel column chromatography (CHCl3/
137.7, 169.9, 175.2, 220.2. ½ꢁꢄ
þ8.8ꢂ (c 0.5, CHCl3). IR
D
(film) cmꢀ1: 3300, 2926, 2097, 1735, 1655, 1625, 1458, 1269, 1185,
752. HRMS m=z (M þ Hþ) Calcd. for C18H25N4O4: 361.1870, Found:
361.1864.
CH3OH = 120/1–40/1) to give
a
mixture of (þ)-5 and its
23
(ꢀ)-15. ½ꢁꢄ
ꢀ9.0ꢂ (c 0.5, CHCl3).
diastereomer (7.40 mg, 20.6 mmol, 63% in 2 steps) as a colorless oil.
A mixture of (þ)-5 and its diastereomer (20.5 mg) was separated by
D
HPLC on
a
triacontyl column (Develosil RPAQUEOUS,
(ꢀ)-14. NMR ꢀH (CDCl3): 1.19 (1H, dd, J ¼ 4:6, 9.6 Hz), 1.25 (1H,
20 ꢃ 250 mm, Nomura Chemical, Nagoya) with 56% aqueous CH3OH
containing 30 mM HCO2NH4 at 8.0 ml/min. Each fraction (2 mg 10
times, (þ)-5; Rt = 24.0–24.5 min, diastereomer (þ)-15; Rt =
24.5–25.0 min) was collected and concentrated in vacuo to give azide
coronatine (þ)-5 (10.3 mg) as a colorless oil and diastereomer (þ)-15
(10.0 mg) as a colorless oil, respectively. The corresponding enan-
tiomers, (ꢀ)-5 and (ꢀ)-15, were obtained by the same procedure using
(þ)-14 instead of (ꢀ)-14.
m), 1.40 (1H, m), 1.71–1.86 (2H, m), 2.00 (2H, brs), 3.15–3.25 (2H,
m), 5.17 (2H, s), 7.27–7.41 (5H, m), ꢀC: 22.7, 26.8, 29.9, 40.1, 51.3,
23
67.1, 128.4, 128.6, 135.7, 174.3. ½ꢁꢄ
ꢀ14ꢂ (c 0.5, CHCl3). IR
D
(film) cmꢀ1
: 3357, 2098, 1720, 1303, 1255, 1153. HRMS m=z
(M þ Hþ) Calcd. for C13H17N4O2: 261.1346, Found: 261.1353.
23
(þ)-14. ½ꢁꢄ
þ15ꢂ (c 0.5, CHCl3).
D
8. NMR ꢀH (CDCl3): 1.11 (1H, t, J ¼ 7:4 Hz), 2.22–2.36 (2H, m),
2.83 (1H, d, J ¼ 7:4 Hz), 3.28 (1H, dd, J ¼ 1:0, 7.4 Hz), 3.96–4.06
(2H, m), 4.16–4.20 (2H, m), 5.15 (1H, dd, J ¼ 1:0, 2.0 Hz), 5.40 (1H,
s), 6.05 (1H, dd, J ¼ 1:0, 2.0 Hz), ꢀC: 11.0, 24.6, 47.6, 50.6, 60.2, 74.0,
(þ)-5. NMR ꢀH (CDCl3): 0.99 (3H, t, J ¼ 7:2 Hz), 1.07 (1H, dd,
J ¼ 12:0, 12.8 Hz), 1.30–1.43 (2H, m), 1.52 (1H, m), 1.55–1.65 (4H,
m), 1.84–1.98 (2H, m), 2.18 (1H, m), 2.25–2.51 (4H, m), 3.14 (1H, m),
3.46 (2H, q, J ¼ 6:0 Hz), 6.37 (1H, s), 6.46 (1H, s), ꢀC: 11.3, 22.7,
76.0, 76.9, 107.7, 127.8, 145.3, 172.5, 186.4, 201.9. IR (film) cmꢀ1
:
25.9, 26.8, 27.8, 28.0, 29.4, 36.1, 37.3, 37.9, 38.1, 46.3, 50.9, 135.3,
3421, 2967, 2938, 2878, 1766, 1677, 1589, 1344, 1250, 1183, 1120,
1041. HRMS m=z (M þ Hþ) Calcd. for C14H17O6: 281.1020, Found:
281.1033.
23
137.7, 169.9, 175.2, 220.2. ½ꢁꢄ
þ69ꢂ (c 0.5, CHCl3). IR
D
(film) cmꢀ1: 3300, 2926, 2097, 1735, 1655, 1625, 1458, 1269, 1185,
752. HRMS m=z (M þ Hþ) Calcd. for C18H25N4O4: 361.1870, Found:
361.1871.
9. NMR ꢀH (CDCl3): 1.11 (1H, t, J ¼ 7:4 Hz), 2.22–2.30 (2H, m),
2.73 (1H, dd, J ¼ 2:2, 7.4 Hz), 3.43 (1H, dd, J ¼ 2:0, 7.4 Hz), 3.68
(1H, brs), 5.21 (1H, dd, J ¼ 2:0, 2.2 Hz), 6.06 (1H, dd, J ¼ 2:0,
2.2 Hz), 6.37 (1H, dd, J ¼ 2:0, 5.9 Hz), 7.75 (1H, dd, J ¼ 2:2, 5.9 Hz),
ꢀC: 11.0, 24.6, 49.1, 49.9, 128.4, 138.8, 145.8, 162.8, 173.9, 204.2. IR
(film) cmꢀ1: 3406, 2966, 2931, 1752, 1709, 1582, 1459, 1344, 1300,
1238, 1193, 1135, 999, 929, 889. HRMS m=z (M þ Hþ) Calcd. for
C14H17O6: 221.0804, Found: 221.0829.
23
(ꢀ)-5. ½ꢁꢄ
ꢀ72ꢂ (c 0.5, CHCl3).
D
(þ)-15. NMR ꢀH (CDCl3): 0.99 (3H, t, J ¼ 7:2 Hz), 1.07 (1H, dd,
J ¼ 12:0, 12.8 Hz), 1.30–1.43 (2H, m), 1.52 (1H, m), 1.55–1.65 (4H,
m), 1.84–1.98 (2H, m), 2.18 (1H, m), 2.25–2.51 (4H, m), 3.14 (1H, m),
3.46 (2H, q, J ¼ 6:0 Hz), 6.37 (1H, s), 6.45 (1H, s), ꢀC: 11.3, 22.4,
25.9, 26.8, 27.8, 28.0, 29.4, 36.1, 37.3, 37.9, 38.1, 46.3, 50.9, 135.3,

2094
M. OKADA et al.
Scheme 1. Syntheses of (þ)-Azido-Coronatine (5) and FITC-Labeled Coronatine (17).
FITC-labeled coronatine (17). To a solution of (þ)-5 (1.10 mg,
single product. Protected azido-coronamic acid ((ꢀ)-14)
can be prepared with ease according to our previous
procedures,9) using (þ)-(R)-4-azido-1,2-butanediol18) as
starting material instead of (þ)-(R)-1,2-butanediol. After
coupling between (ꢁ)-13 and (ꢀ)-14, followed by
HPLC purification, (þ)-azido-COR (5) was obtained in
10 steps with 11% yield with its diastereomer (þ)-15
(10%).19) The enantiomer of (þ)-5 ((ꢀ)-ent-5) and its
diastereomer ((ꢀ)-ent-15) were obtained similarly.
The stereochemistry of (þ)-5 and (ꢀ)-ent-5 was
assigned by a comparison of the ½ꢁꢄD value together
with the results of bioassay. A comparison of ½ꢁꢄD
3.05 mmol), FITC-alkyne 16 (2.40 mg, 3.25 mmol), and sodium ascor-
bate (643 mg, 3.25 mmol) in EtOH (330 ml) and 1 M aqueous NaHCO3
(330 ml) was added CuSO4 (104 mg, 0.652 mmol). After stirring for 12 h,
the reaction mixture was quenched with 5% aqueous KHSO4. This
mixture was extracted with EtOAc (4 ꢃ 3 ml), and the resulting
organic layer was washed with saturated aqueous NaCl, dried over
Na2SO4, and filtered. After evaporation, the residue was purified by
silica gel column chromatography (CHCl3/CH3OH ¼ 40/1–10/1) to
give 1616) (3.30 mg, 3.00 mmol, 89%) as a yellowish solid. NMR ꢀH
(CD3OD): 0.78–0.91 (4H, m), 1.08–1.43 (3H, m), 1.68 (1H, m), 1.92–
2.28 (14H, m), 3.41–3.87 (7H, m), 6.15 (1H, d, J ¼ 16:0 Hz), 6.35 (1H,
s), 6.42–6.45 (2H, m), 6.56–6.60 (4H, m), 7.06 (1H, d, J ¼ 8:2 Hz),
7.67 (1H, d, J ¼ 9:6 Hz), 8.03 (1H, s), 8.37 (2H, s), 8.64 (1H, s).
HRMS m=z (M þ Hþ) Calcd. for C54H48F6N7O10S: 1100.3082, Found:
1100.3090.
23
23
23
values among (þ)-5 (½ꢁꢄ
þ69ꢂ), (ꢀ)-ent-5 (½ꢁꢄ
D
D
23
ꢀ72ꢂ), (þ)-15 (½ꢁꢄ
þ8.8ꢂ), and (ꢀ)-ent-15 (½ꢁꢄ
D
D
ꢀ9.0ꢂ) strongly suggested that (þ)-5 corresponds to the
naturally occurring stereoisomer of COR. Moreover,
(þ)-5 was as effective as naturally occurring (þ)-2 in
the stomatal opening assay using I. tricolor, whereas
(ꢀ)-ent-5 was not effective in the same assay (Fig. 2).
Moreover, since (þ)-azide-COR (5) was verified to
maintain the bioactivity of original 1 (Fig. 2), it can be
used as a platform for chemical probes.
Results and Discussion
The synthesis of azide-COR (5) is described in
Scheme 1. We achieved on improvement in product
yield in the synthesis of (ꢁ)-coronafacic acid by
examination of the synthetic route. For example, exo-
selectivity in the Diels-Alder reaction was improved to
endo:exo > 1:25 by the use of 6 as substrate,17) and the
yields in the subsequent reductions were also improved
by the use of 8 or ꢁ,ꢂ-unsaturated ketone (9) as
intermediate. Fortunately, the reduction of 9 in toluene
proceeded with high stereoselectivity to give (S)-10 as a
Easy operation using click chemistry should provide
molecular probes with various tags fitting any purposes,
such as fluorescence-dye for detection of bindingsites,
biotin for chemiluminescence detection, and epitope
peptides for immuno precipitation. The resulting 5

Synthesis of Azido-Coronatine
2095
References and Note
HO2
C
HO2C
N3
O
H
NH
O
H
NH
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This work was supported by Grants-in-Aid for
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