(3R,7S)-12-Hydroxy-jasmonoyl-l-isoleucine is the genuine bioactive stereoisomer of a jasmonate metabolite in Arabidopsis thaliana.

The oxylipin plant hormone (3R,7S)-jasmonoyl-l-isoleucine [or (+)-7-iso-jasmonoyl-l-isoleucine, JA-Ile] is widely recognized as a plant defense hormone against pathogens and chewing insects. The metabolism of JA-Ile into 12-OH-JA-Ile and 12-COOH-JA-Ile is the central mechanism for the inactivation of JA signaling. Recently, 12-OH-JA-Ile was reported to function as a ligand for the JA-Ile co-receptor COI1-JAZ. However, in previous studies, '12-OH-JA-Ile' used was a mixture of four stereoisomers, the naturally occurring cis-isomer (3R,7S)-12-OH-JA-Ile and the trans-isomer (3R,7R)-12-OH-JA-Ile, and the unnatural cis-isomer (3S,7R)-12-OH-JA-Ile and the trans-isomer (3S,7S)-12-OH-JA-Ile. Thus, the genuine bioactive form of 12-OH-JA-Ile has not yet been identified. In the present study, we prepared pure stereoisomers of 12-OH-JA-Ile and identified (3R,7S)-12-OH-JA-Ile as the naturally occurring bioactive form of 12-OH-JA-Ile and found that it binds to COI1-JAZ9 as effectively as (3R,7S)-JA-Ile. In addition, we revealed that the unnatural trans-isomer (3S,7S)-12-OH-JA-l-Ile functions as another bioactive isomer. The pure (3R,7S)-12-OH-JA-Ile causes partial JA-responsive gene expression without affecting the expression of JAZ8/10, which is involved in the negative feedback regulation of JA-signaling. Thus, (3R,7S)-12-OH-JA-Ile could cause weak and sustainable expression of certain JA-responsive genes until the catabolism of (3R,7S)-12-OH-JA-Ile into (3R,7S)-12-COOH-JA-Ile occurs. The use of chemically pure (3R,7S)-12-OH-JA-Ile confirmed the genuine biological activities of '12-OH-JA-Ile' by excluding the possible effects of other stereoisomers. A chemical supply of pure (3R,7S)-12-OH-JA-Ile with an exact bioactivity profile will enable further detailed studies of the unique role of 12-OH-JA-Ile in planta.

Subtype-selective agonists of plant hormone co-receptor COI1-JAZs identified from the stereoisomers of coronatine.

Severe genetic redundancy is particularly clear in gene families encoding plant hormone receptors, each subtype sharing redundant and specific functions. Genetic redundancy of receptor family members represents a major challenge for the functional dissection of each receptor subtype. A paradigmatic example is the perception of the hormone (+)-7-iso-jasmonoyl-L-isoleucine, perceived by several COI1-JAZ complexes; the specific role of each receptor subtype still remains elusive. Subtype-selective agonists of the receptor are valuable tools for analyzing the responses regulated by individual receptor subtypes. We constructed a stereoisomer library consisting of all stereochemical isomers of coronatine (COR), a mimic of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine, to identify subtype-selective agonists for COI1-JAZ co-receptors in Arabidopsis thaliana and Solanum lycopersicum. An agonist selective for the Arabidopsis COI1-JAZ9 co-receptor efficiently revealed that JAZ9 is not involved in most of the gene downregulation caused by COR, and the degradation of JAZ9-induced defense without inhibiting growth.

Host-Selective Phytotoxins Incorporating the Epoxy-Triene-Decacarboxylate Moiety Function through the Hijacking of the Plant-Microbe Interaction System.

Host selective toxins (HSTs) are small molecule phytotoxins that control the pathogenicity of microbes in the host plant, but the mechanistic basis for their selectivity is unknown. We developed AcIle-EDA (Aclle-(+)-9,10-epoxy-8-hydroxy-9-methyldeca-trienoic acid) as a molecular probe of an HST, examined its mode of action in genetically modified Oryza sativa, and found it to trigger ROS production through NADPH-oxidase OsRBOHB, causing the emergence of pathogenic traits. This result strongly suggests that AcIle-EDA functions through the hijacking of the plant-microbe interaction system.

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15(Z)-phytodienoic acid (OPDA), and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5Z,8Z)-10-(4-oxo-5-((Z)-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ4-dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia. By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha.

Deciphering OPDA Signaling Components in the Momilactone-Producing Moss Calohypnum plumiforme.

Jasmonic acid (JA) and its biologically active form jasmonoyl-L-isoleucine (JA-Ile) regulate defense responses to various environmental stresses and developmental processes in plants. JA and JA-Ile are synthesized from α-linolenic acids derived from membrane lipids via 12-oxo-phytodienoic acid (OPDA). In the presence of JA-Ile, the COI1 receptor physically interacts with JAZ repressors, leading to their degradation, resulting in the transcription of JA-responsive genes by MYC transcription factors. Although the biosynthesis of JA-Ile is conserved in vascular plants, it is not recognized by COI1 in bryophytes and is not biologically active. In the liverwort Marchantia polymorpha, dinor-OPDA (dn-OPDA), a homolog of OPDA with two fewer carbons, and its isomer dn-iso-OPDA accumulate after wounding and are recognized by COI1 to activate downstream signaling. The moss Calohypnum plumiforme produces the antimicrobial-specialized metabolites, momilactones. It has been reported that JA and JA-Ile are not detected in C. plumiforme and that OPDA, but not JA, can induce momilactone accumulation and the expression of these biosynthetic genes, suggesting that OPDA or its derivative is a biologically active molecule in C. plumiforme that induces chemical defense. In the present study, we investigated the biological functions of OPDA and its derivatives in C. plumiforme. Searching for the components potentially involving oxylipin signaling from transcriptomic and genomic data revealed that two COI1, three JAZ, and two MYC genes were present. Quantification analyses revealed that OPDA and its isomer iso-OPDA accumulated in larger amounts than dn-OPDA and dn-iso-OPDA after wounding. Moreover, exogenously applied OPDA, dn-OPDA, or dn-iso-OPDA induced the transcription of JAZ genes. These results imply that OPDA, dn-OPDA, and/or their isomers potentially act as biologically active molecules to induce the signaling downstream of COI1-JAZ. Furthermore, co-immunoprecipitation analysis showed the physical interaction between JAZs and MYCs, indicating the functional conservation of JAZs in C. plumiforme with other plants. These results suggest that COI1-JAZ-MYC mediated signaling is conserved and functional in C. plumiforme.

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.

Stereoselective Syntheses of all the Possible Stereoisomers of Coronafacic Acid.

An efficient and stereoselective syntheses of all the possible stereoisomers of coronafacic acid (CFA) has been developed. The stereochemistries of C3a and C7a were controlled in a diastereoselective Diels-Alder type cycloaddition using a chiral auxiliary. CFA and 6-epi-CFA were synthesized by hydrogenation of a common intermediate. During the synthesis of 6-epi-CFA, we established that its cis-fused configuration is important for the introduction of C4-C5 double bond by dehydration. This report is the first practical synthesis of both 6-epi-CFA, and its enantiomer.

A scalable synthesis of (+)-coronafacic acid.

A facile, efficient, and scalable synthesis of optically pure coronafacic acid by resolution of racemic coronafacic acid obtained using an improved version of Watson's method has been developed. By optimizing the boron-mediated aldol reaction of Watson, we were able to prepare 2.1 g of racemic coronafacic acid. This was coupled with (S)-4-isopropyl-2-oxazolidinone to give a mixture of diastereomeric coronafacyl oxazolidinones, which were readily separable by silica-gel column chromatography to give 630 mg of optically pure (+)-coronafacic acid.

Methylene chain ruler for evaluating the regioselectivity of a substrate-recognising oxidation catalyst.

Regioselective C-H oxidation of aliphatic molecules with synthetic catalysts is challenging. We incorporated substrate-recognition sites into a ruthenium porphyrin-heteroaromatic N-oxide catalytic system in order to characterise its regioselectivity for the oxidation of alkanes. This substrate-recognition catalytic reaction exhibits high regioselectivity and high reaction efficiency.

Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate on Spectroscopy and Catalytic Activity.

We synthesized intramolecularly aliphatic alcoholate-coordinated iron porphyrins (1a, 1b) that retain their axial coordination in the presence of another ligand or oxidant. The electron-donative character of alcoholate was less than that of thiolate, and the coordination ability of a sixth ligand to 1a and 1b was very much lower than in the case of the thiolate-coordinated compounds. Density functional theory calculations indicated that the marked difference in coordination ability could be explained in terms of thermodynamic and steric factors. The catalytic oxidizing ability of the thiolate-coordinated compound, SR complex, was much higher than that of 1a.

A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants.

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein-protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy-based in vitro binding assay system to screen for the ligands of the COI1-JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader-based assays of both agonists and antagonists of COI1-JAZ co-receptors.

Effect of the o-Acetamido Group on pH-Dependent Light Emission of a 3-Hydroxyphenyl-Substituted Dioxetane Luminophore.

A pioneering chemiluminescent molecule reported by Schaap and co-workers, 3-(2'-spiroadamantane)-4-methoxy-4-(3″-hydroxy)phenyl-1,2-dioxetane (AMPD), does not require enzymatic activation but is unsuitable for use under physiological conditions. To overcome this limitation, we have developed a new AMPD derivative that contains an acetamido group at the ortho position of the hydroxy group as an intramolecular hydrogen-bonding site in order to lower the p Ka value. This compound exhibits a superior chemiluminescence response to AMPD in the physiologically relevant pH range.

Potent Antimalarial Activity of Two Arenes Linked with Triamine Designed To Have Multiple Interactions with Heme.

Based on the idea that compounds designed to exhibit high affinity for heme would block hemozoin formation, a critical heme-detoxification process for malarial parasites, we synthesized a series of compounds with two π-conjugated moieties at terminal amino groups of triamine. These compounds exhibited moderate to high antimalarial activities in vitro toward both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. In a P. berghei-infected mouse model, 3a and 12a showed potent antimalarial activities compared to artesunate, as well as a prolonged duration of antimalarial effect. We found a good correlation between protective activity against hemin degradation and antimalarial activity. Compounds 8b and 3a strongly inhibited hemozoin formation catalyzed by heme detoxification protein.

A rationally designed JAZ subtype-selective agonist of jasmonate perception.

The phytohormone 7-iso-(+)-jasmonoyl-L-isoleucine (JA-Ile) mediates plant defense responses against herbivore and pathogen attack, and thus increases plant resistance against foreign invaders. However, JA-Ile also causes growth inhibition; and therefore JA-Ile is not a practical chemical regulator of plant defense responses. Here, we describe the rational design and synthesis of a small molecule agonist that can upregulate defense-related gene expression and promote pathogen resistance at concentrations that do not cause growth inhibition in Arabidopsis. By stabilizing interactions between COI1 and JAZ9 and JAZ10 but no other JAZ isoforms, the agonist leads to formation of JA-Ile co-receptors that selectively activate the JAZ9-EIN3/EIL1-ORA59 signaling pathway. The design of a JA-Ile agonist with high selectivity for specific protein subtypes may help promote the development of chemical regulators that do not cause a tradeoff between growth and defense.

A small-molecule inhibitor of SOD1-Derlin-1 interaction ameliorates pathology in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder. Despite its severity, there are no effective treatments because of the complexity of its pathogenesis. As one of the underlying mechanisms of Cu, Zn superoxide dismutase (SOD1) gene mutation-induced ALS, SOD1 mutants (SOD1mut) commonly interact with an endoplasmic reticulum-resident membrane protein Derlin-1, triggering motoneuron death. However, the importance of SOD1-Derlin-1 interaction in in vitro human model and in vivo mouse model remains to be elucidated. Here, we identify small-molecular-weight compounds that inhibit the SOD1-Derlin-1 interaction by screening approximately 160,000 compounds. The inhibitor prevents 122 types of SOD1mut from interacting with Derlin-1, and significantly ameliorates the ALS pathology both in motoneurons derived from patient induced pluripotent stem cells and in model mice. Our data suggest that the SOD1-Derlin-1 interaction contributes to the pathogenesis of ALS and is a promising drug target for ALS treatment.

Jasmonic Acid Inhibits Auxin-Induced Lateral Rooting Independently of the CORONATINE INSENSITIVE1 Receptor.

Plant root systems are indispensable for water uptake, nutrient acquisition, and anchoring plants in the soil. Previous studies using auxin inhibitors definitively established that auxin plays a central role regulating root growth and development. Most auxin inhibitors affect all auxin signaling at the same time, which obscures an understanding of individual events. Here, we report that jasmonic acid (JA) functions as a lateral root (LR)-preferential auxin inhibitor in Arabidopsis (Arabidopsis thaliana) in a manner that is independent of the JA receptor, CORONATINE INSENSITIVE1 (COI1). Treatment of wild-type Arabidopsis with either (-)-JA or (+)-JA reduced primary root length and LR number; the reduction of LR number was also observed in coi1 mutants. Treatment of seedlings with (-)-JA or (+)-JA suppressed auxin-inducible genes related to LR formation, diminished accumulation of the auxin reporter DR5::GUS, and inhibited auxin-dependent DII-VENUS degradation. A structural mimic of (-)-JA and (+)-coronafacic acid also inhibited LR formation and stabilized DII-VENUS protein. COI1-independent activity was retained in the double mutant of transport inhibitor response1 and auxin signaling f-box protein2 (tir1 afb2) but reduced in the afb5 single mutant. These results reveal JAs and (+)-coronafacic acid to be selective counter-auxins, a finding that could lead to new approaches for studying the mechanisms of LR formation.

Role of Thiolate Ligand in Spin State and Redox Switching in the Cytochrome P450 Catalytic Cycle.

The catalytic cycle of cytochrome P450 involves a change from the resting-state, water-bound, six-coordinated form (1, low-spin state) to a five-coordinated form (2, high-spin state) upon binding of a hydrophobic substrate. Here, we used a heme-thiolate model complex (SR complex) with THF as a model of nonionic H2O to address the question of whether or not coordination of nonionic water is sufficient to induce the low-spin state. Measurements of electronic absorption spectra and magnetic properties confirmed that five-coordinated SR complex has a high-spin state, and THF-bound, six-coordinated SR has a low-spin state in dichloromethane at ambient temperature. The redox potential E1/2 (FeII/FeIII) of THF-bound SR was 80-90 mV more negative than that of five-coordinated SR. These properties indicate SR is a good model of P450. Our results suggest that thiolate coordination plays a key role in setting the low energy barrier between the high-spin and low-spin states.

Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

[Tuberculous epididymitis after Bacillus Calmette-Guérin intravesical therapy : a case report].

We describe a case of tuberculous epididymitis following Bacillus Calmette-Guérin (BCG) instillation in a 79-year-old man. He had received transuretheral resection of bladder tumor in Sep 2009. Histopathological diagnosis was urothelial carcinoma, high grade, pTa and pTis. To prevent recurrence, he received maintenance therapy for Feb 2010-May 2011 after eight weekly intravesical instillation of BCG. Skin fistula with discharge in the left scrotum occurred in Sep 2011. Treatment with levofloxacin was not effective. Therefore, we performed bilateral orchiectomy (he had hormone therapy of prostate cancer) and left scrotal skin resection. Histopathological examine showed tuberculous epididymitis. He had no signs of recurrence 2 years postoperatively and has not received any anti-tuberculous chemotherapy.

Nitrous oxide reduction-coupled alkene-alkene coupling catalysed by metalloporphyrins.

Porphyrin complexes of iron, manganese and cobalt catalysed reductive dimerization of alkenes with NaBH4 under N2O. The reaction system using Fe(III) porphyrin generated an Fe(I) porphyrin intermediate that reduced N2O to regenerate Fe(III) porphyrin.