Syntheses of dinor-cis/iso-12-oxo-phytodienoic acid (dn-cis/iso-OPDAs), ancestral jasmonate phytohormones of the bryophyte Marchantia polymorpha L., and their catabolites

In recent years, the biology of the evolutionary origin of phytohormone signaling has made significant progress. Among them, the ligand-receptor co-evolution found in jasmonate signaling has attracted the attention of plant scientists. Dinor-cis-12-oxo-phytodienoic acid (dn-cis-OPDA, 4) and dn-iso-OPDA (5) are ancestral plant hormones of the bryophyte Marchantia polymorpha L. We succeeded in the first practical synthetic supply of these hormones as well as their possible catabolites. These compounds are expected to be useful in the study of ancestral jasmonate signaling in bryophytes.


Results and discussion
Synthesis of dn-cis-OPDA (4) and its potent catabolites (6 and 7). Our plan for the synthesis of dn-cis-OPDA (4) and its catabolites (6 and 7) is outlined in Scheme 1. A major initial concern in the syntheses of 4, 6 and 7 was the avoidance of epimerization at C11 which was anticipated to be facile in the presence of either acid or base based on the ready epimerization of the structurally similar 1 to give the more thermodynamically stable trans-1 in a ratio of trans:cis = 95:5 21 . We therefore planned to synthesize 4 according to a procedure similar to that used to synthesize OPDA (10), a congener of 4, developed by Kobayashi et al. 22 . They avoided epimerization by introducing the ketone at the late stage of synthesis 23 . Compounds 4 and 6 would be obtained from a common intermediate 11 by Wittig reaction using a different phosphonium salt (Scheme 1), and 7 would be obtained by oxidation of 6. www.nature.com/scientificreports/ Our synthesis of dn-cis-OPDA (4) is summarized in Scheme 2. Allylic substitution of monoacetate 15, prepared by enzymatic reaction 22 , with TBDPS(CH 2 ) 6 MgCl, was performed in the presence of CuCN to afford 16. The Mitsunobu reaction of 16, hydrolysis of resulting acetate and Eschenmoser-Claisen rearrangement gave dimethylamide 17. Iodolactonization, elimination with DBU and subsequent reduction with LiAlH 4 afforded diol 18. In the iodolactonization step, use of water in place of buffer resulted in the removal of TBDPS group. TES Scheme 1. Synthetic plan for dn-cis-OPDA (4) and its potent catabolites (6 and 7). Scheme 2. Synthesis of dn-cis-OPDA (4). Reagents and conditions: (a) TBDPSO(CH 2 ) 6  In the work up of final Jones oxidation, removal of chromium compounds and sulfuric acid using silica gel caused epimerization of C11 (probably due to exothermic adsorption of sulfuric acid on silica gel) 22 , but this could be easily avoided by removal of the inorganic substances with water instead. The obtained 4 was quite stable at room temperature under neutral condition and no epimerization at C11 was observed even after 2 weeks [25][26][27] .

Synthesis of dn-iso-OPDA (5) and its potent catabolites (8 and 9).
Our plan for the synthesis of dn-iso-OPDA (5) and its potent catabolites (8 and 9) is shown in Scheme 4. In the synthesis of [ 2 H 2 ]-tetrahydrodicanenone (iso-OPDA), Lauchli and Boland Introduced the C1-8 side chain by the 1,4-addition using an organozinc reagent and CuCN 28 . However, organozinc reagent are difficult to prepare and CuCN is highly toxic. In contrast, Grignard reagents used for the 1,2-addition are easy to prepare and less toxic than CuCN. And potent catabolites 16-OH-dn-iso-OPDA (8) and 16-COOH-dn-iso-OPDA (9) could be obtained from the same starting material 24 by using a different allyl bromide.
Our synthesis of dn-iso-OPDA (5) is summarized in Scheme 5. Allylation of 1,3-cyclopentanedione 24 followed by methylation of the resulting 27 gave cyclopentenone 28. After the Grignard reaction of 28, dilution of the reaction mixture with hydrochloric acid promoted hydrolysis of the enol ether and the deprotection of the THP group, to give alcohol 30. Finally, Jones oxidation of 30 gave 61 mg of dn-iso-OPDA (5) in only 4 steps from 24.

Conclusions
A synthetic supply of jasmonates is indispensable if their study is to be advanced 20,30,33,34 , and this work finally enables dn-cis/iso-OPDAs (4 and 5) and their potent catabolites (6-9) to be readily obtained. Our work is expected to accelerate biological studies of the signaling mechanisms of bryophyte hormones, which should lead to a better understanding of the evolutional origins of phytohormone signaling. In particular, study of the catabolism of 4 and 5 should provide insights into the deactivation mechanism of ancestral plant hormones. Biological studies using synthetic 4-9 are now in progress.  To a solution of diol intermediate (15.3 mg, 60.6 µmol) in acetone (5.2 mL) was added Jones reagent (4.0 M solution) at − 20 °C until the orange color of the reagent persisted (30 drops). After 10 min of stirring at − 20 °C, i-PrOH was added to quench the remaining reagent. Then, EtOAc/n-hexane (1/1, 20 mL) and H 2 O (20 mL) were added and the water layer was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by medium-pressure chromatography (Isolera, eluent: 0.1:88:12 AcOH/n-hexane/EtOAc to 0.1:99.9 AcOH/ EtOAc) to give 4 (116 mg, 97%) as a colorless oil. Diastereomeric purity of 4 was > 99% by 1     After 20 min of stirring at − 20 °C, i-PrOH was added to quench the remaining reagent. Then, EtOAc (10 mL) and H 2 O (60 mL) were added and the water layer was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by medium-pressure chromatography (Isolera, eluent: 0.1:98:2 AcOH/CHCl 3 /MeOH to 0.1:80:20 AcOH/CHCl 3 /MeOH) to give 7 (8.3 mg, 73%) as a colorless oil. Diastereomeric purity of 7 was > 98% by 1  was added a solution of 28 (198 mg, 1.10 mmol) in THF (6.5 mL) at reflux temperature under argon atmosphere. After being stirred at 60 °C for 3 h, the reaction mixture was allowed to cool to rt and 2 M HCl aq. (7 mL) was added. After 1.5 h of stirring, H 2 O was added and the water layer was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4 and concentrated under reduced pressure. After evaporation, the residue was purified by medium-pressure chromatography (Isolera, eluent: 40:60 n-hexane/EtOAc to EtOAc) to give the oxidized compound. The compound was carried on to the next step.
To a solution of the mixture (79.6 mg) in acetone (6 mL) was added Jones reagent (4.0 M solution, 200 µL, 800 µmol) at 0 °C. After 3.5 h of stirring at 0 °C, i-PrOH was added to quench the remaining reagent. Then, H 2 O (20 mL) was added and the water layer was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by medium-pressure chromatography (Isolera, eluent: 0.5:50:50 AcOH/n-hexane/EtOAc to 0.5:99.5 AcOH/ EtOAc) to give 5 (61.4 mg, 89%) as a colorless oil. 1