BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 8 potx

8
Coronatine: Chemistry and Biological Activities
Akitami Ichihara and Hiroaki Toshima
CONTENTS
8.1 Introduction
8.2 Structures of Coronatine and Analogs
8.3 Biosynthesis of Coronatine
8.4 Syntheses of Coronatine and Biosynthetic Analogs of Jasmonic Acid
8.4.1 Asymmetric Total Synthesis of Coronatine
8.4.2Synthesis of Analogs of Jasmonic Acid and its Biosynthetic
Intermediates
8.5 Biological Activities of Coronatine
8.5.1 Allocoronamic Acid as Inhibitors of Ethylene Biosynthesis
8.5.2 Similar Biological Activities of Coronatine to Those of Jasmonic Acid
8.6 Perspective
References
8.1 Introduction
The phytotoxin, coronatine (1), was isolated from the phytopathogenic bacterium
Pseudomonas syringae pv. atropurpurea which causes chocolate spot disease on the leaves of
Italian ryegrass.
1
After structural determination of 1, it was found that several pathovars of
Pseudomonas syringae and Xanthomonas campestris pv. phormiicola produce 1 as a phytotoxic
compound. Closely related analogues of 1 were isolated from Pseudomonas syringae pv. gly-
cinea, pv. tomato, and Xanthomonas campestris pv. phormiicola. Recently, there has been much
interest in 1, since various biological activities similar to those of jasmonic acid were shown
in assays for tuber-inducing activity, etc. Furthermore, the potent activities of 1 are 100 to
10,000 times higher than those of jasmonic acid. Therefore, 1 is the most valuable probe in
the area of plant physiological studies relating to jasmonic acid. In order to supply a suffi-
cient amount of 1, an efficient synthetic method is urgently needed, since the productivity
of 1 by P. syringae pathovars is rather fluctuated depending on the culture conditions

and/or mutation. The total synthesis of optically active coronatine (1) was accomplished
and made it possible to supply a practical amount of (+)-1. Analogues of 1 and jasmonic
acid, and the compounds related to biosynthetic intermediates and amino acid conjugates
of jasmonic acid were also synthesized and their biological activities were evaluated in
some bioassay systems. In addition to the hormonal activities of coronatine, the similarity
© 1999 by CRC Press LLC
of the structure of coronamic acid with that of 1-amino-1-cyclopropanecarboxylic acid
(ACC), the direct precursor of the plant hormone ethylene, made it possible to develop
inhibitors of ethylene biosynthesis.
8.2 Structures of Coronatine and Analogs
The structure and stereochemistry of coronatine (1) were determined by spectroscopic
methods and x-ray analyses of coronafacic acid (2) and (–)-N-acetylcoronamic acid.
2-4
The
structure of coronatine (1) is quite unique in that it consists of coronafacic acid (2) and coro-
namic acid (3) (Figure 8.1). After the discovery of 1, it was found that several P. syringae
pathovars (pv. maculicola,
5
pv. glycinea,
6
pv. morsprunorum
7
and pv. tomato
8
) produce 1.
Besides coronatine, Pseudomonas syringae pv. glycinea produces several coronatine ana-
logues, norcoronatine (4), N-coronafacoyl-L-valine (5), N-coronafacoyl-L-isoleucine (6),
and N-coronafacoyl-L-alloisoleucine (7) which are all biologically active.
9,10
The absolute

configuration of norcoronatine (4) was confirmed by the comparison of physicochemical
data with those of natural and synthetic specimens.
11
Furthermore, Pseudomonas syringae
pv. tomato produces N-caronafacoyl-L-valine (5)
12
and Xanthomonas campestris pv. phormii-
cola produces N-coronafacoyl-L-valine (5) and N-coronafacoyl-L-isoleucine produces (6).
13
It also was proved that Xanthomonas campestris pv. phormiicola produces coronatine (1)
itself.
4
8.3 Biosynthesis of Coronatine
The two components of coronatine (1) are biosynthesized from two different pathways
respectively (Scheme 8.1). Coronafacic acid (2) was shown to be a novel polyketide derived
FIGURE 8.1
Structures of (+)-coronatine and the related compounds.
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from three acetate units, one butyrate unit, and one unit of pyruvate.
15
Recently, it was
found that the labeled pyruvate was carboxylated to oxaloacetate which was further con-
verted into α-ketoglutarate through the TCA cycle. Further support of the intermediacy of
α-ketoglutarate was provided by incorporation experiments of a labeled precursor,
[3,4-
13
C
2
]-DL-glutamate to give a labeled 2 at C-2 and C-3. Interestingly, Mitchell et al.
16

have
described the isolation of the cyclopentenone derivative 8 from Pseudomonas syringae pv.
tomato which is able to produce coronatine as a major component. Feeding experiments of
the [3,4-
13
C
2
]-DL-glutamate into this bacterium gave a labeled cyclopentenone 8 which
exhibits
13
C-
13
C coupling between C-2 and C-3 with an enrichment at each carbon of
ca. 0.89%. The coupling pattern is completely consistent with that observed in coronafacic
acid (2). The results also are compatible with the previous biosynthetic studies of
coronafacic acid, and the cyclopentenone 8 would be a plausible intermediate that would
be transformed by Michael’s addition of malonyl CoA to an indanone derivative 8a and
successive decarboxylation, reduction of the C-5 carbonyl, and dehydration to give
coronafacic acid (2) (Scheme 8.2).
The biosynthetic studies of coronamic acid (3) showed that both L-isoleucine and L-alloi-
soleucine are specifically incorporated into the amino acid (3), the latter L-alloisoleucine is
a much more efficient precursor. Since the absolute configuration of coronamic acid (3) is
consistent with that of L-alloisoleucine, the incorporation efficiency is easily understand-
able, though the cyclization mechanism has been completely elucidated.
17
Previously, coro-
natine analogues, N-coronafacoyl-L-isoleucine (6) and N-coronafacoyl-L-alloisoleucine,
were regarded as promising intermediates in the biosynthesis of coronatine (1). However,
this is not the case. Since biosynthetic condensation of coronafacic acid (2) and coronamic
acid (3) has been made by the incorporation of

14
C-coronamic acid (3) into coronatine (1) in
Pseudomonas syringae pv. glycinea PG 4180.
18
Extensive genetic studies have been done with
the same strain and, using genetic complementation, substrate feeding, and co-culturing
experiments, regions involved in the biosynthesis of major intermediates, coronafacic acid
(2) and coronamic acid (3) and the ligase of these components were identified in the coro-
natine production gene cluster.
19
SCHEME 8.1
Biosynthesis of (+)-coronatine.
SCHEME 8.2
Plausible biosynthesis of (+)-coronafacic acid.
© 1999 by CRC Press LLC
8.4 Syntheses of Coronatine and Biosynthetic Analogs
of Jasmonic Acid
8.4.1 Asymmetric Total Synthesis of Coronatine
We have reported the partial synthesis of (+)-coronatine (1) by coupling between the acid chlo-
ride (2c) derived form natural (+)-coronafacic acid (2) and synthetic (+)-coronamic acid (3) as
a free amino acid.
3
This route is not efficient because the optical resolution of racemic 3 is
required and the coupling yield is low (see Scheme 8.5). In order to supply a sufficient amount
of (+)-1, we have developed a new route via the asymmetric syntheses of (+)-2 and (+)-3.
We chose a most straightforward route for (+)-3 involving tandem alkylation of the mal-
onate anion with a dielectrophile derived from (R)-1, 2-butanediol (9) (Scheme 8.3).
20
(R)-Malic acid was used as a chiral source and converted to 9 in 52% of overall yield in five
steps. By treating 9 with thionyl chloride and then ruthenium tetraoxide, cyclic sulfate 10

was quantitatively obtained. Dibenzyl malonate anion, generated with 2 eq. of sodium
hydride, was alkylated with 10 to give the cyclopropane derivative 11 in 93% yield. The
first intermolecular substitution occurred at a primary site to give a b-sulfate which was
smoothly cyclized to 11 by intramolecular substitution with inversion of the stereogenic
center. Alkaline hydrolysis of 11 gave the half-acid 12 in 91% yield and 96% d.e., because
the sterically less-hindered ester was selectively hydrolyzed. Treatment of 12 with diphe-
nylphosphoryl azide (DPPA) by refluxing in tert-butanol gave the protected amino acid
derivative 13 via Curtius rearrangement in 76% yield. Recrystallization of 13 from ethyl
acetate/hexane increased the purity. One pot (alkaline and subsequent acidic) hydrolysis
of 13, ion exchange, and crystallization from acetone/H
2
O gave (+)-3 (89% yield, >99.8%
e.e., 96.6% d.e.); mp > 190°C (sublimation); [α]
D
23
+ 15.8° (c 1.10, H
2
O) [lit. [α]
D
20
+ 14.7°
(c 1.67, H
2
O)]. Of course, 13 can be used as an amine component for amide formation after
SCHEME 8.3
Syntheses of all four stereoisomers of coronamic acid.
© 1999 by CRC Press LLC
removal of the Boc group under usual acidic conditions. Furthermore, esterification of 12
with diazomethane and subsequent hydrogenolysis gave another half-acid 14 which could
be converted to (+)-allocoronamic acid [(+)-16]. Antipodes, (–)-coronamic acid [(–)-3] and

(–)-allocoronamic acid [(–)-16], were synthesized starting from (S)-malic acid. In this way,
we could develop a practical route for all four stereoisomers of coronamic acid in high
chemical yields and high optical purities without the optical resolution step. Several grams
of the four isomers were synthesized by our new route.
We have already developed a new approach using intramolecular 1,6-conjugate addition
for construction of a 1-hydrindane framework such as 2 on a racemic substrate
(Scheme 8.4).
21
By this methodology, the C
6
-non, C
6
-methyl, and C
6
-butyl substituted ana-
logs [(±)-19b, -19c, -19d, -20a, -20b, -20c, and -20d] of 2, could be synthesized from (±)-18a-d
(Scheme 8.4).
22
In principle, this method would be applied to synthesize the optically active
form.
23,24
For our synthesis of (+)-2, the efficient preparation of the optically active ester
[(+)-17] is required. This problem was solved using the catalytic asymmetric Michael reac-
tion promoted by the heterobimetallic BINOL complex developed by Shibasaki and co-
workers.
25
Michael reaction between 2-cyclopenten-1-one and diethyl malonate was car-
ried out in the presence of Ga-Na-(S)-BINOL complex (10 mol%) and sodium tert-butoxide
(7 mol%) to give a keto-diester in 95% yield. Acetalization and subsequent decarboxylation
gave desired the ester (+)-17 in 74% yield. The optical purity of (+)-17 was determined as

95% e.e. by an HPLC analysis using a chiral column which was sufficiently pure for subse-
quent reactions. The total synthesis of (+)-2 was carried out according to the synthetic route
of racemic 2. Aldol condensation between the lithium enolate of (+)-17 and 2-ethylacrolein
proceeded stereospecifically in the newly formed stereogenic centers to give a ca. 1:1 mix-
ture of 21a and 21b which possesses a syn-relationship between the ethoxycarbonyl and
hydroxyl groups. Mesylation of the mixture of 21a and 21b and subsequent β-elimination
with DBU gave α, β, δ, γ-unsaturated ester possessing (E)-geometry as the sole product.
Further deacetalization with p-TsOH in acetone gave (–)-18a, the precursor of intramolec-
ular 1,6-conjugate addition in 78% yield [four steps from (+)-17]. Treatment of (–)-18a with
NaOEt (3 eq.) in EtOH gave 22a corresponding to the ethyl ester of coronafacic acid and its
C
6
-epimer 22b in 71% yield (diastereoselectivity; ca. 3:1). Isomerization of 22b with DBA
also was possible to give a 1:1 mixture of 22a and 22b. The optical purity of 22a was deter-
mined as 98% e.e. by an HPLC analysis using a chiral column. This result demonstrates that
the optical purity of (+)-17 was completely retained in 22a without any loss through the
sequence of reactions including intramolecular 1,6-conjugate addition. The effectiveness of
our new approach, on both racemic and optically active forms, was proven. Acidic hydrol-
ysis of 22a gave (+)-2 in 70% yield after recrystallization; mp 142-143°C; [α]
D
23
+122° (c 1.00,
MeOH) [lit. mp 141-142°C; [α]
D
20
+119° (c 3.30, MeOH)], whose spectral data were identical
with those of natural 2 in all respects. The overall yield via our new route was 24% in nine
steps from 2-cyclopenten-1-one. The optical purity of (+)-2 is estimated to be at least >98%
e.e. based on 98% e.e. of 22a. In practice, judging from the specific rotation, (+)-2 can be
regarded as optically pure.

After deprotection of the Boc group of 13 with TFA, the resulting amine TFA salt 13a
(evaporated to dryness and used without purification) was coupled with (+)-2 in the pres-
ence of a water-soluble carbodiimide to give coronatine benzyl ester (1a) in 89% yield
(Scheme 8.5). In a preliminary experiment, coupling between racemic 2 and optically active
13a gave a mixture of 1a and its diastereomer (inseparable on TLC), whose
1
H-NMR spec-
trum gave partially separated signals based on the respective diastereomers (ratio ca. 1:1).
However, in the case of coupling (+)-2, the
1
H-NMR spectrum of 1a was observed as the sin-
gle diastereomer. This result means that the synthetic (+)-2 has practical enantiomeric purity
and 1a can be regarded as optically pure. Deprotection of 1a by hydrogenolysis in the pres-
ence of 10% Pd-C in ethyl acetate provided (+)-1 in 80% yield; mp 162-164°C [α]
D
22
+76.6°
© 1999 by CRC Press LLC
SCHEME 8.4
Syntheses of coronafacic acid and its analogs.
© 1999 by CRC Press LLC
(c 2.20, MeOH) [lit. mp 161-163°C; [α]
D
20
+68.4° (c 2.20, MeOH)]. The spectral data of syn-
thetic (+)-1 were identical with those of natural 1 in all respects including the specific rota-
tion. In this way, the first asymmetric total synthesis of (+)-1 has been accomplished. The
yield was remarkably improved in contrast to the previous synthesis of (+)-1 from natural 2.
8.4.2 Syntheses of Analogs of Jasmonic Acid
and its Biosynthetic Intermediates

Even-numbered OPC homologs (OPC-8:0, -6:0, and -4:0) as biosynthetic precursors of jas-
monic acid (JA) (Scheme 8.6) and odd-numbered OPC homologues (OPC-7:0, -5:0, -3:0, and
SCHEME 8.5
Total synthesis of coronatine.
SCHEME 8.6
Biosynthesis of jasmonic acid and structural similarity to coronatine.
© 1999 by CRC Press LLC
-1:0) were synthesized from 2-[(Z)-2-pentenyl]cyclopenten-1-one (23) as the common starting
material in short steps and with high yields via conjugate addition (Scheme 8.7).
26
Only in
the case of synthesizing OPC-1:0, tris(phenylthio)methyl lithium as a C
1
-synthon was used
for the conjugate addition to give the orthothioester which was converted to OPC-1:0 (24a)
via ethanolysis and subsequent hydrolysis. In the other cases, zinc–copper reagents which
were prepared from iode-esters possessing the requisite carbon chain, were applied for the
conjugate addition. The resulting esters were hydrolyzed to OPC homologues [OPC-3:0
(24b), -4:0 (24c); -5:0 (24d), -6:0 (24e), -7:0 (24f), and -8:0 (24g)]. Each acid was observed as
a trans-rich mixture in the
13
C-NMR spectra.
Esters of odd-numbered OPC homologues (25a, 25b, 25d, and 25f) are convenient sub-
strates for synthesizing analogs which are not structurally subject to β-oxidation.
27
Protec-
tion of the carbonyl group and subsequent LAH reduction gave an alcohol which was
treated with tert-butyl bromoacatate to give a 3-oxa-OPC ester. Acidic hydrolysis gave
3-Oxa-OPC-8:0 (26d), -6:0 (26b), and -4:0 (26a). Instead of LAH reduction, DIBAL reduction
gave an aldehyde which was treated with trichloroacetic acid/sodium trichloroacetate to

give a trichloromethylcarbinol. Fluorination and hydrolysis gave 2-fluoro-OPC-8:0 (27f),
-6:0 (27d), and -4:0 (27b). Each acid also was observed as trans-rich mixture in the
13
C-NMR
spectra.
N-(Jasmonyl)amino acid conjugates (JA-AAs) were also synthesized from JA (racemic
and trans-rich) and the appropriate amino acids.
28
In addition to naturally occurring JA-
Leu, JA-Ile, JA-Val (derived from aliphatic amino acids), JA-Phe, JA-Tyr, and JA-Trp
(derived from aromatic amino acids), not naturally occurring JA-Gly, JA-β-Ala, JA-ACC,
JA-(+)-coronamic acid were also synthesized. Among them, JA-Leu, JA-Ile, JA-Val,
JA-ACC, and JA-(+)-coronamic acid are structurally similar to coronatine.
SCHEME 8.7
Syntheses of OPC-homologous series, 3-oxa-OPC- and 2-fluoro-OPC-analogs.
© 1999 by CRC Press LLC
8.5 Biological Activities of Coronatine
8.5.1 Allocoronamic Acid as Inhibitors of Ethylene Biosynthesis
Although naturally occurring coronamic acid [(+)-3] has the (1S, 2S)-configuration, three
other possible stereoisomers [(–)-3, (+)-16, and (–)-16, see Scheme 8.3] are considered and
have been prepared via optical resolution or asymmetric synthesis as described above.
3,20,29
Coronamic acids are regarded as analogs of ACC and correspond to substituted ACC with
an ethyl group. ACC is well known as the direct precursor of a plant hormone, ethylene.
Therefore, coronamic acids were applied to elucidating stereospecific recognition of ACC
by the ethylene-forming enzyme (EFE).
30
One stereoisomer, (1R, 2S)-allocoronamic acid
[(+)-16] has been found to be converted to 1-butene at a 50 to 250 times higher rate than any
of the other stereoisomers (Scheme 8.8). Since 1-butene does not exhibit ethylene-like

actions, (+)-16 would be used as a competitive inhibitor of ethylene biosynthesis. There-
fore, we examined the inhibitory effect of coronomic acids on senescence in cut carnation
flowers (Dianthus caryophyllus L. cv. Lightpink Barbara) (Table 8.1).
31
Although both race-
mic and optically active allocoronamic acids exhibited antisenescent activity, (+)-16 exhib-
ited the strongest activity at 10 mM (longevity, 12 to 13 days). The longevity was slightly
shorter than that of silver thiosulfate complex (STS) used as a positive control. In the case
of using (–)-16, the stems changed to yellow and broke in extreme cases. In addition, we
measured the relative EFE activity using a tomato suspension-cell culture. The relative EFE
activity was decreased by both (+)-16 (78.2%) and (–)-16 (85.8%). α-aminoisobutyric acid
(AIB) which is a known competitive inhibitor of EFE, also decreased the relative EFE activ-
ity with similar intensity (84.7%) to those of allocoronamic acids. Thus, it was proven that
allocoronamic acids acted as inhibitors of ethylene biosynthesis.
SCHEME 8.8
Biosynthesis of ethylene and stereospecific recognition of ACC/(+)-allocoronamic acid by EFE.
© 1999 by CRC Press LLC
8.5.2 Similar Biological Activities of Coronatine to Those of Jasmonic Acid
A chlorosis-inducing phytotoxin, 1, was isolated with the guidance of the potato cell
expansion-inducing activity, and the hormone-like activities of 1 already have been stud-
ied. Recently, there has been strong interest in 1 which has been shown to exhibit various
biological activities similar to those of JA, known as an endogenous plant-growth regulator
and signal transmitter.
32
JA and JA-Me (or as jasmonoid) are recognized as a kind of phy-
tohormone and known to exhibit various biological activities in higher plants: senescence-
promoting activity, growth-inhibitory activity, potato tuber-inducing activity, potato cell
expansion-inducing activity, etc. These biological activities also are induced by 1 and the
latter two activities were the first discovery in the similarity of the biological activities of
coronatine to those of JA. Furthermore, the potent activities of 1 are 100 to 10,000 times

higher than those of JA.
33-35
There are other examples that revealed that 1 and JA (or JA-Me)
exhibited similar biological activities. Both 1 and JA-Me caused similar growth-inhibitory
effects on Arabidopsis (seedling and root), stimulated anthocyanin accumulation, and
increased the level of two proteins (31 and 29 kD). Arabidopsis mutants selected for resis-
tance to 1 are male sterile are also insensitive to JA-Me and resistant to bacterial patho-
gens.
36
It was proven with a tomato suspension culture that both 1 and JA played a central
role in regulating EFE activity in ethylene biosynthesis.
33,34
Both 1 and JA stimulated the
biosynthesis and emission of volatiles (terpenes, acetogenins, and aromatics) in many plant
species.
37
Antitumor taxane-type diterpenes were increased ca. 10 times by treating Taxus
baccta with 1 and JA-Me.
38
In the tendril coiling assay of Bryonia dioica, 1, the 12-oxo-PDA
methyl ester, the OPC-8:0 methyl ester, and JA-Me exhibited activity that decreased in that
order.
39
This result suggested the importance of not only JA, but octadecanoid precursors as
signaling molecules of higher plants. In our recent study (Figures 8.2 and 8.3), the following
results were obtained: (1) JA-amino acid conjugate (JA-Leu, JA-Ile, JA-Val) exhibited weaker
TABLE 8.1
Inhibitory Effect of Coronamic Acids on Senescence
in Cut Carnation Flowers (Dianthus caryophyllus L.
cv. Lightpink Barbara)

Concentration Longevity
Compound
a
(mM) (Days)
Control (H
2
O) – 6
STS (Koto fresh K-20) 1/1000
c
15
(±)-CA 10 6
(+)-(1S,2S)-CA 5 6–7
(–)-(1R,2R)-CA 5 6
(±)-AlloCA 10 8–10
(±)-AlloCA
b
10 10–11
(+)-(1R,2S)-AlloCA 10 12–13
5 10–11
1.67 9
(–)-(1S,2R)-AlloCA 10 8–9
5 9–10
1.67 10–11
a
Three flowers were used for each compound tested.
b
After treating the flowers in the reagent for 24 h, the
solution was changed to distilled water.
c
The original solution of a commercially available STS

reagent was used after diluting 1000 times.
© 1999 by CRC Press LLC
FIGURE 8.2
Structure–activity relationship in potato cell expansion-inducing assay.
FIGURE 8.3
Structure–activity relationship in potato tuber-inducing assay.
© 1999 by CRC Press LLC
potato cell expansion-inducing activity than that of JA; (2) OPC-8:0, -6:0, and -4:0 exhibited
both potato cell expansion-inducing and tuber-inducing activity at a similar concentration
to JA; (3) not naturally occurring OPC-7:0 and OPC-5:0 also exhibited tuber-inducing activ-
ity; (4) the C6-alkyl-substituted analogs of 2 exhibited weak tuber-inducing activity;
22
(5) a
unique activity, decreasing the weight of potato tissue by 2-fluoro-OPC-8:0 in the cell
expansion-inducing assay was first detected; and (6) 3-oxa-OPC-8:0, -6:0, and -4:0 also
exhibited weak tuber-inducing activity. The last result suggests that β-oxidation is not
required and that the corresponding biosynthetic precursor of JA, OPC-8:0, -6:0, and -4:0 in
themselves exhibit tuber-inducing activity. In all the examined results of comparative bio-
assays of 1, JA, biosynthetic precursors and analogs, 1 was always the most active com-
pound. Since in vivo coronatine has not been isolated as a secondary product, 12-oxo-PDA
and/or OPC-homologs would play an important role as signaling compounds in higher
plants.
40-44
8.6 Perspective
From the first isolation of coronatine as a phytotoxin, about 20 years have already past.
During the period, coronatine was applied to various disciplinary fields and played impor-
tant roles. There has been strong interest in coronatine again with respect to jasmonic acid-
like activities. At the present stage, coronatine is the most promising probe for investigating
plant physiological actions relating to JA (jasmonoid and octadecanoid). The elucidation of
the JA-(jasmonoid- and octadecanoid-) receptor family is the remaining biggest problem,

and coronatine would play an important role in solving the problem. Our asymmetric syn-
thesis is able to provide a certain amount of coronatine; however, further synthetic studies
on more structurally simple and biologically active analogs of coronatine should continue,
and might result in developing a new plant-regulatory agent. The synthetic approach
becomes increasingly important to obtain a practical, marketable product.
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