Verification of the versatility of the in vitro enzymatic reaction giving (+)-cis-12-Oxo-phytodienoic acid

Article abstract of DOI:10.1016/j.bmcl.2021.128284

Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic pathway of arabidopsides could occur via MGDG.

Full text of DOI:10.1016/j.bmcl.2021.128284

Bioorg. Med. Chem. Lett. 49 (2021) 128284
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Bioorganic & Medicinal Chemistry Letters
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Verification of the versatility of the in vitro enzymatic reaction giving
(+)-cis-12-Oxo-phytodienoic acid
,
,
,*
Yusuke Itoa 1, Kento Sasakia 1, Tsuyoshi Ogiharaa, Naoki Kitaokaa, Kosaku Takahahib
,
Hideyuki Matsuuraa
,
*
a Laboratory of Natural Product Chemistry, Division of Fundamental AgriScience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589,
Japan
b Department of Nutritional Science, Faculty of Applied BioScience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
A B S T R A C T
Jasmonic acid (JA) is a plant hormone involved in the defense response against insects and fungi. JA is synthesized from α-linolenic acid (LA) by the octadecanoid
pathway in plants. 12-oxo-Phytodienoic acid (OPDA) is one of the biosynthetic intermediates in this pathway. The reported stereo selective total synthesis of cis-
(+)-OPDA is not very efficient due to the many steps involved in the reaction as well as the use of water sensitive reactions. Therefore, we developed an enzymatic
method for the synthesis of OPDA using acetone powder of flax seed and allene oxide cyclase (PpAOC2) from Physcomitrella patens. From this method, natural cis-
(+)-OPDA can be synthesized in the high yield of approximately 40%. In this study, we investigated the substrate specificity of the enzymatic synthesis of other OPDA
analogs with successions to afford OPDA amino acid conjugates, dinor-OPDA (dn-OPDA), and OPDA monoglyceride, and it was suggested that the biosynthetic
pathway of arabidopsides could occur via MGDG.
Varieties of JA-amino acid conjugates, such as JA-L-Ile, JA-L-Gly, JA-
L-Ala, JA-L-Leu, JA-L-Val, JA-L-Phe, JA-L-Tyr, and JA-L-Trp, have been
reported 1. We speculated that there would also be different types of
OPDA analogs in nature; in fact, OPDA-L-Ile was isolated from Arabi-
dopsis thaliana 2. In investigating the other conjugates, it would be
helpful to use the authentic compound as an indicator for purification
and identification. In a previous paper, an efficient enzymatic synthetic
method yielding (9S,13S)-12-oxo-phytodienoic acid (OPDA) was re-
ported by Kajiwara et al. 3, and the reported method was characterized
by the reaction solution containing acetone powder prepared from flax
seeds 4 and a recombinant AOC derived from Physcomitrella patens 5. The
OPDA yield from the reaction using this system was almost 7-fold higher
than that obtained from the conventional reaction with flaxseed extract
4 and gave a compound with an absolute configuration that is consistent
with that of natural OPDA. Moreover, OPDA-L-Ile was shown to be
Phe, L-Tyr, L-Trp, L-Glu, and L-Gln, Figure 1), whose spectroscopic data
are given in the Supplementary data. Then, all synthesized compounds
were subjected to the method described in the previously reported paper
3 to give OPDA conjugated with L-Gly, L-Ala, L-Val, L-Leu, L-Ile, L-Phe, L-
Tyr, L-Trp, L-Glu, and L-Gln. The reactions gave the expected compounds
(Figure 1), whose spectroscopic data are given in the Supplementary
data, although the conversion of LA-L-Trp into OPDA-L-Trp either did not
proceed at all or did not proceed to produce an amount of compound
that could withstand instrumental analysis. The synthetic yields of
OPDA-L-Glu and OPDA-L-Gln were 38% and 48%, respectively, and the
yields of OPDA-L-Gly (26%), OPDA-L-Ala (24%), OPDA-L-Val (25%),
OPDA-L-Leu (15%), OPDA-L-Ile (14%), and OPDA-L-Phe (16%), and
OPDA-L-Tyr (4%) were lower than those of OPDA-L-Glu and OPDA-L-Gln
(Figure 2). The reason why the reaction yield of OPDA-L-Ile in this paper
(14%) is lower than the one in the reference paper (35%) 6 is probably
due to the fact that the conversion capacity of the enzyme obtained from
flax seeds used in the reaction cannot be kept constant each time. One of
the most likely reasons for the inconsistency may be the quality of the
flax seeds. Notably, the reaction using LA-L-Trp as a starting material did
not give the expected compound. In general, it was thought that starting
materials with high hydrophilicity, such as LA-L-Glu and LA-L-Gln, gave
better yields and that compounds conjugated with larger amino acid
biosynthesized from an isoleucine conjugate of α-linolenic acid in Ara-
bidopsis thaliana 6. It was therefore hypothesized that this method could
be applied to synthesize OPDA-amino acid conjugates and OPDA ana-
logs such as dinor-OPDA ((7R,11S)-dn-cis-OPDA) and 2,3-dihydroxy-
propyl 12-oxo-phytodienoate (OPDA monoglyceride).
As a first step, we synthesized 10 types of α-linolenic acid (LA)-amino
acid conjugates (LA conjugated with L-Gly, L-Ala, L-Val, L-Leu, L-Ile, L-
* Corresponding authors.
E-mail addresses: kt207119@nodai.ac.jp (K. Takahahi), matsuura@chem.agr.hokudai.ac.jp (H. Matsuura).
1
The authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128284
Received 11 May 2021; Received in revised form 9 July 2021; Accepted 19 July 2021
Available online 23 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

Y. Ito et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128284
Figure 1. Chemical structures of the LA and OPDA amino acid conjugates.
Figure 2. Enzymatic conversion to give (7R,11S)-dn-cis-OPDA.
molecules with lower hydrophilicity, such as L-Trp, were unsuitable for
the reaction.
however no expected compounds were detected in the case of the re-
action using LA monoglyceride. (Figure 3A). This unexpected result was
thought to be due to hydrolysis caused by the activity of lipase in the
enzyme solution containing the acetone powder originating from flax
seeds. Therefore, an attempt was made to detect the compound using
UPLC MS/MS, which is able to detect trace amount of compounds. After
synthesizing authentic OPDA monoglyceride, whose synthetic proced-
ure is given in supplementary data and optimizing the parameters to
detect the monoglyceride, a portion of the reaction mixture using LA
monoglyceride as a substrate was subjected to UPLC MS/MS. The fea-
tures of the UPLC MS/MS chromatographs are given in Figure 3B, and it
was found that the reaction using LA monoglyceride gave OPDA
monoglyceride based on the compound from the reaction mixture and
the authentically synthesized OPDA monoglyceride having the same
retention time. This result revealed that the system using acetone
powder of flax seed and allene oxide cyclase was able to carry out the
series of reactions of LOX, AOS, and AOS and accept LA monoglyceride
as a substrate. In order to suggest the hypothesis that the giving trace
amount of OPDA may be due to the lipase activity in the reaction
mixture, we tried to detect OPDA in the reaction solution after
attempting the conversion of an LA monoglyceride. The features of the
UPLC MS/MS chromatograph are given in Figure 3C, and OPDA was
detected as expected. However, it might also be possible that OPDA,
which originated from the LA released from LA monoglyceride after
lipase treatment, formed an ester bond with the liberated propane-1,2,3-
triol to give OPDA monoglyceride. In order to verify this possibility, the
method reported by Kajiwara et al. 3 was performed with some modi-
fications, in which OPDA and propane-1,2,3-triol were added as sub-
strates instead of LA monoglyceride. The UPLC MS/MS data is shown in
Figure 3D, indicating that the esterification of OPDA with propane-
1,2,3-triol did not proceed, whose result ruled out a reaction mecha-
nism in which OPDA is formed and then combined with propane-1,2,3-
triol to produce OPDA-monoglyceride.
In 2018, Monte et al. 7 reported that (7R,11S)-dn-cis-OPDA 8 was a
crucial component that regulates defense, growth, and developmental
responses in Marchantia polymorpha, and it was assumed that the re-
ported method to obtain OPDA from LA should be applicable to give
(7R,11S)-dn-cis-OPDA from (7Z,10Z,13Z)-hexadeca-7,10,13-trienoic
acid. (7Z,10Z,13Z)-Hexadeca-7,10,13-trienoic acid was isolated from
radish leaves (Raphanus sativus) (2 kg) according to the procedure
described in the Supplementary data, and the isolated compound was
subjected to the reaction, yielding the expected compound (7R,11S)-dn-
cis-OPDA (Figure 2) in a conversion rate of 23%, whose spectroscopic
data given in the Supplementary data coincided well with the reported
those of synthetic (7R,11S)-dn-cis-OPDA 9. However, the optical rotation
23
([
α]
+ 110.5) of (7R,11S)-dn-cis-OPDA synthesized in this study was
D
23
lower than that of reported one ([
α
]
+ 135.5) 9. The reason for the
smaller optical rotation was considereDd to be the isomerization of the cis
isomer in the process of isolating the compound, although the contam-
ination of trans isomer was not judged from 1H NMR spectrum. Wang
et al. reported that a typical resonance for cis isomer is δH 7.72 (dd, J =
5.8, 2.7 Hz, 1H), whereas that for trans isomer is δH 7.59 (dd, J = 5.8,
2.6 Hz, 1H).
Arabidosides are well known characteristic secondary metabolites of
A. thaliana. Arabidosides are presumed to be a metabolite derived from
monogalactosyldiacylglycerol (MGDG), but it was also conceivable that
the compounds might be biosynthesized in the series of reaction, in
which OPDA might bind to propane-1,2,3-triol. The detailed biosyn-
thetic pathway that affords arabidosides has not yet been clarified,
although it has been suggested that fatty acids remain attached to gal-
actolipids during the enzymatic conversion to give OPDA 10. The syn-
thesis of LA monoglycerides was accomplished according to a reported
method 11, whose spectroscopic data are given in the Supplementary
data, and the obtained LA monoglyceride was then subjected to the re-
action described in the report of Kajiwara et al. 3 We could detect the
expected compounds by TLC analysis in the case of former reactions,
In order to substantiate the above mentioned conclusion, the appli-
cation of (9Z,12Z,15Z)-N-(2,3-dihydroxypropyl)octadeca-9,12,15-
2

Y. Ito et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128284
Figure 3. Enzymatic conversion of LA mono-glyceride to OPDA mono-glyceride. A) Enzymatic conversion of LA-monoglyceride to OPDA mono-glyceride (m/z 366)
and OPDA (m/z 290). B) Detection of OPDA mono-glyceride using UPLC MS/MS in positive mode. Upper column for the reaction mixture. Lower column for
authentic sample, C) Detection of OPDA in the reaction mixture using UPLC MS/MS in negative mode, D) Detection of OPDA mono-glyceride using UPLC MS/MS in
positive mode. Upper column for the reaction mixture. Lower column for authentic sample. x): indicating pseudo molecular and transition ions to detect OPDA mono-
glyceride. y): indicating pseudo molecular and transition ions to detect OPDA.
trienamide, which has an amide bond that is less susceptible to lipase
degradation than the ester bond, was carried out (Figure 4). The syn-
thesis of (9Z,12Z,15Z)-N-(2,3-dihydroxypropyl)octadeca-9,12,15-
trienamide was performed according to the detailed procedure in the
Supplementary data, and the synthesized substrate was subjected to the
reaction according to the method described in the literature 3, which
gave enough amount of N-(2,3-dihydroxypropyl)-
Fig. 4B and spectroscopic data given in the Supplementary data.
Because OPDA monoglycerides could be enzymatically synthesized
from 2,3-dihydroxypropyl linoleate (LA monoglyceride), this result
could support the story that LOX, AOS, and AOC act upon MGDG to
afford arabidosides in nature, similar to the conclusion reported by
Nilsson et al. 10 Proposed biosynthetic pathway to give arabidopside via
Path A are given in Fig. 5 together with experimentally concreated
biosynthetic pathway to afford (+)-7-isoJA and (-)-JA.
8-((1S,5S)-4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)octana-
mide (24% synthetic yield) (Fig. 4A) to withstand detection using TLC
and the 1H NMR measurements, whose feature of 1H NMR are given in
In the present study, the enzymatic method for the synthesis of OPDA
developed by Kajiwara et al. 3 was found to be applicable to synthesize
3

Y. Ito et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128284
Figure 4. Enzymatic conversion to give N-(2,3-dihydroxypropyl)-8-((1S,2S)-3-oxo-2- ((Z)-pent-2-en-1-yl) cyclopentyl)octanamide. A) Chemical structures of
(9Z,12Z,15Z)-N-(2,3-dihydroxypropyl) octadeca-9,12,15- trienamide and N-(2,3-dihydroxypropyl)-8-((1S,5S)-4-oxo-5-((Z)-pent-2-en-1- yl)cyclopent-2-en-1-yl)
octanamide. B) 1H NMR spectrum of N-(2,3-dihydroxypropyl)-8- ((1S,5S)-4-oxo-5-((Z)-pent-2-en-1-yl) cyclopent-2-en-1-yl)octanamide (270 MHz, CDCl3).
OPDA analogs such as OPDA-amino acid conjugates, (7R,11S)-dn-cis-
OPDA, and OPDA-monoglyceride. These results indicated that there was
loose substrate recognition of the carboxylic acid moiety of fatty acids by
the biosynthetic enzymes. As mentioned above, A. thaliana synthesizes a
series of unique compounds, arabidopsides A-E. Arabidopside A pro-
motes senescence of barley leaves (Hordeum vulgare) 12, and arabidop-
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
14
sides A, B, and D inhibit the elongation of roots 13. Anderson et al.
Acknowledgments
reported that arabidopside E showed an inhibitory effect on the growth
of Pseudomonas syringae in vitro. The proteins AvrRpm1 and AvrRpt2 are
attenuated proteins derived from P. syringae, and it has been reported
that arabidopside E accumulation occurs when these two proteins are
recognized by Arabidopsis thaliana 15. However, the biosynthetic
We acknowledge the assistance of Dr. Eri Fukushi and Mr. Yusuke
Takata (Research Faculty of Agriculture, Hokkaido University) in
obtaining the spectroscopic data. We used UPLC MS/MS systems at the
Research Faculty of Agriculture, Hokkaido University. This work was
supported by the Japan Society for the Promotion of Science (JSPS)
[KAKENHI Grant Number 20H04755 to HM].
pathway that produces these compounds have not been completely
10
uncovered. Anders et al.
reported that fatty acids could remain
attached to galactolipids during the enzymatic conversion to (dn) OPDA,
and our present report supports this experimental result, in which the
conversion of MGDG into arabidopsides proceeds while the fatty acids
are bound to the galactolipids. However, in order to give a true picture of
the biosynthesis of arabidopsides, further studies are needed.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128284.
4

Y. Ito et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128284
Figure 5. Biosynthetic pathway to afford arabidopside and jasmonic acid. Chemical structures of a representative MGDG, bis-α linolenic acid form (a) and repre-
sentative arabidoside, arabidoside B (b). cis-OPDA: cis-12-oxo-phytodienoic acid, OPC 8:0: 3-oxo-2-(2′-[Z]-pentenyl)-cyclopentane-1-octanoicacid, OPC 6:0; 3-oxo-2-
(2′- [Z]-pentenyl)-cyclopentane-1-hexanoic acid, OPC 4:0: 3-oxo-2-(2′-[Z]-pentenyl)- cyclopentane-1-butanoicacid. LOX: lipoxygenase, AOS: alene oxide synthase,
AOC: alene oxide cyclase, OPR3: cis-12-oxophytodienoate reductase 3, JA: jasmonic acid.
9 Wang JX, Sakurai H, Kato N, Kaji T, Ueda M. Syntheses of dinor-cis/iso-12-oxo-
References
phytodienoic acid (dn-cis/iso-OPDAs), ancestral jasmonate phytohormones of the
bryophyte Marchantia polymorpha L., and their catabolites. Sci Rep. 2021;11:2033.
1 Staswick P, Tiryaki I. The oxylipin signal jasmonic acid is activated by an enzyme
10 Nilsson AK, Fahlberg P, Ellerstrom M, Andersson MX. Oxo-phytodienoic acid (OPDA)
that conjugates it to isoleucine in Arabidopsis. Plant Cell. 2004;16(8):2117–2127.
is formed on fatty acids esterified to galactolipids after tissue disruption in
2 Flokova K, Feussner K, Herrfurth C, et al. A previously undescribed jasmonate
Arabidopsis thaliana. FEBS Lett. 2012;586(16):2483–2487.
compound in flowering Arabidopsis thaliana - The identification of cis-(+)-OPDA-Ile.
11 Ogihara T, Amano N, Mitsui Y, et al. Determination of the Absolute Configuration of
Phytochem. 2016;122:230–237.
a Monoglyceride Antibolting Compound and Isolation of Related Compounds from
3 Kajiwara A, Abe T, Hashimoto T, Matsuura H, Takahashi K. Efficient Synthesis of (
Radish Leaves (Raphanus sativus). J Nat Pro. 2017;80(4):872–878.
+)-cis-12-Oxo-phytodienoic Acid by an in Vitro Enzymatic Reaction. Biosci Biotech
12 Hisamatsu Y, Goto N, Hasegawa K, Shigemori H. A glycolipid involved in flower bud
Biochem. 2012;76(12):2325–2328.
formation of Arabidopsis thaliana. Bot Stud. 2006;47(1):45–50.
4 Zimmerman DC, Feng P. Characterization of a prostaglandin-like metabolite of
13 Hisamatsu Y, Goto N, Sekiguchi M, Hasegawa K, Shigemori H. Oxylipins
linolenic acid produced by a flax-seed extract. Lipids. 1978;13(5):313–316.
arabidopsides C and D from Arabidopsis thaliana. J Nat Pro. 2005;68(4):600–603.
¨
5 Stumpe M, Gobel C, Faltin B, et al. The moss Physcomitrella patens contains
14 Andersson MX, Hamberg M, Kourtchenko O, et al. Oxylipin profiling of the
cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to
hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-
reduced fertility and altered sporophyte morphology. New Phytol. 2010;188(3):
phytodienoic acid-containing galactolipid, arabidopside E. J Biol Chem. 2006;281
740–749.
(42):31528–31537.
6 Uchiyama A, Yaguchi T, Nakagawa H, et al. Biosynthesis and in vitro enzymatic
¨
¨
15 Kourtchenko O, Andersson M, Hamberg M, Brunnstrom A, Gobel C, McPhail K,
synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine
¨
Gerwick W, Feussner I, Ellerstrom M, Oxo-phytodienoic acid-containing galactolipids
conjugate of
α
-linolenic acid. Bioorg Med Chem Lett. 2018;28(6):1020–1023.
in Arabidopsis: jasmonate signaling dependence. Plant Physiol 2007;145(4):1658-
1669.
˜
7 Monte I, Ishida S, Zamarreno AM, et al. Ligand-receptor co-evolution shaped the
jasmonate pathway in land plants. Nat Chem Biol. 2018;14(5):480–488.
8 Ogorodnikova AV, Gorina SS, Mukhtarova LS, et al. Stereospecific biosynthesis of
(9S,13S)-10-oxo-phytoenoic acid in young maize roots. Biochim Biophys Acta. 2015;
1851(9):1262–1270.
5