Synthesis and biological activity of photostable and persistent abscisic acid analogs.

The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'-trans-diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.

Ergosterol and Its Metabolites Induce Ligninolytic Activity in the Lignin-Degrading Fungus Phanerochaete sordida YK-624.

White-rot fungi are the most important group of lignin biodegraders. Phanerochaete sordida YK-624 has higher ligninolytic activity than that of model white-rot fungi. However, the underlying mechanism responsible for lignin degradation by white-rot fungi remains unknown, and the induced compounds isolated from white-rot fungi for lignin degradation have never been studied. In the present study, we tried to screen ligninolytic-inducing compounds produced by P. sordida YK-624. After large-scale incubation of P. sordida YK-624, the culture and mycelium were separated by filtration. After the separation and purification, purified compounds were analyzed by high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance. The sterilized unbleached hardwood kraft pulp was used for the initial evaluation of ligninolytic activity. Ergosterol was isolated and identified and it induced the lignin-degrading activity of this fungus. Moreover, we investigated ergosterol metabolites from P. sordida YK-624, and the ergosterol metabolites ergosta-4,7,22-triene-3,6-dione and ergosta-4,6,8(14),22-tetraen-3-one were identified and then chemically synthesized. These compounds significantly improved the lignin-degrading activity of the fungus. This is the first report on the ligninolytic-inducing compounds produced by white-rot fungi.

Photostable Abscisic Acid Agonists with a Geometrically Rigid Cyclized Side Chain.

The plant hormone abscisic acid (ABA) plays a central role in adaptive responses to abiotic stresses that adversely affect crop growth and productivity. However, ABA photoinstability limits its use in agriculture. To overcome this drawback, in this study, we developed photostable ABA analogues, the (+)-BP2A compound series (compounds 5-9), in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. All BP2A analogues showed higher stability against UV-B irradiation at 302 nm than ABA, and compounds 6 and 7 barely decomposed even under sunlight. In physiological assays, (+)-BP2A and (+)-compound 7, in which the α,ß-unsaturated carbonyl group of BP2A was reduced, exhibited ABA-like activities, including inhibition of seed germination and induced drought tolerance in Arabidopsis. Biochemical studies revealed that (+)-compound 7, unlike (+)-BP2A, did not activate pyrabactin resistance-like (PYL) receptors in vitro and was converted to (+)-BP2A in plants, suggesting that it functions as a prodrug PYL agonist. Furthermore, (+)-compound 7 inhibited seed germination of tomato, lettuce, and rice. Thus, this compound represents a potential plant growth regulator that induces ABA-type responses in agricultural fields.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

Keeping the shoot above water - submergence triggers antithetical growth responses in stems and petioles of watercress (Nasturtium officinale).

The molecular mechanisms controlling underwater elongation are based extensively on studies on internode elongation in the monocot rice (Oryza sativa) and petiole elongation in Rumex rosette species. Here, we characterize underwater growth in the dicot Nasturtium officinale (watercress), a wild species of the Brassicaceae family, in which submergence enhances stem elongation and suppresses petiole growth. We used a genome-wide transcriptome analysis to identify the molecular mechanisms underlying the observed antithetical growth responses. Though submergence caused a substantial reconfiguration of the petiole and stem transcriptome, only little qualitative differences were observed between both tissues. A core submergence response included hormonal regulation and metabolic readjustment for energy conservation, whereas tissue-specific responses were associated with defense, photosynthesis, and cell wall polysaccharides. Transcriptomic and physiological characterization suggested that the established ethylene, abscisic acid (ABA), and GA growth regulatory module for underwater elongation could not fully explain underwater growth in watercress. Petiole growth suppression is likely attributed to a cell cycle arrest. Underwater stem elongation is driven by an early decline in ABA and is not primarily mediated by ethylene or GA. An enhanced stem elongation observed in the night period was not linked to hypoxia and suggests an involvement of circadian regulation.

Design of potent ABA receptor antagonists based on a conformational restriction approach.

The physiological functions of the plant hormone abscisic acid (ABA) are triggered by interactions between PYR/PYL/RCAR receptors (PYLs) and group-A protein phosphatases 2C (PP2Cs). PYL agonists/antagonists capable of inducing/disrupting these interactions would be valuable in investigating the regulatory mechanisms of ABA signaling. Previously, we developed (+)-PAO4, a high-affinity PYL antagonist, by conformationally restricting the S-hexyl chain of our first reported PYL antagonist, 3'-hexylsulfanyl-ABA. Although (+)-PAO4 shows a greater binding affinity for Arabidopsis PYL5 compared with 3'-hexylsulfanyl-ABA, it is not able to completely block the ABA responses both in vitro and in vivo. Therefore, we designed novel conformationally restricted PYL antagonists in which the O-butyl chain of (+)-PAO4 was replaced with a pentyl (PAC4), a pentyne (PAT3) or a pentadiyne (PATT1) chain. (+)-PAT3 and (+)-PATT1 suppressed the ABA-induced inhibition of Arabidopsis seed germination more strongly than (+)-PAO4, but contrary to expectations, the affinity of each compound for PYL5 was almost the same as that of (+)-PAO4. Subsequent biochemical analyses revealed that unlike (+)-PAO4, (+)-PAT3 and (+)-PATT1 completely abolished ABA-induced PYL-PP2C interactions without partial agonistic activities. The superior PYL antagonist functions of (+)-PAT3 and (+)-PATT1 over (+)-PAO4 may explain their potent antagonistic activities against exogenous ABA in vivo. Furthermore, (+)-PAT3 and (+)-PATT1 also suppressed ABA responses in rice, indicating that both compounds are useful chemical tools for ABA-signaling studies, not only in dicots but also in monocots.

Dynamic control of plant water use using designed ABA receptor agonists.

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.

Structure-Based Chemical Design of Abscisic Acid Antagonists That Block PYL-PP2C Receptor Interactions.

In Arabidopsis, signaling of the stress hormone abscisic acid (ABA) is mediated by PYR/PYL/RCAR receptors (PYLs), which bind to and inhibit group-A protein phosphatases 2C (PP2Cs), the negative regulators of ABA. X-ray structures of several PYL-ABA and PYL-ABA-PP2C complexes have revealed that a conserved tryptophan in PP2Cs is inserted into a small tunnel adjacent to the C4' of ABA in the PYL-ABA complex and plays a crucial role in the formation and stabilization of the PYL-ABA-PP2C complex. Here, 4'-modified ABA analogues were designed to prevent the insertion of the tryptophan into the tunnel adjacent to the C4' of ABA in these complexes. These analogues were predicted to block PYL-PP2C receptor interactions and thus block ABA signaling. To test this, 4'- O-phenylpropynyl ABA analogues were synthesized as novel PYL antagonists (PANs). Structural, thermodynamic, biochemical, and physiological studies demonstrated that PANs completely abolished ABA-induced PYL-PP2C interactions in vitro and suppressed stress-induced ABA responses in vivo more strongly than did 3'-hexylsulfanyl-ABA (AS6), a PYL antagonist we developed previously. The PANs and AS6 antagonized the effects of ABA to different degrees in different plants, suggesting that these PANs can function as chemical scalpels to dissect the complicated regulatory mechanism of ABA signaling in plants.

Abscinazole-E3M, a practical inhibitor of abscisic acid 8'-hydroxylase for improving drought tolerance.

Abscisic acid (ABA) is an essential phytohormone that regulates plant water use and drought tolerance. However, agricultural applications of ABA have been limited because of its rapid inactivation in plants, which involves hydroxylation of ABA by ABA 8'-hydroxylase (CYP707A). We previously developed a selective inhibitor of CYP707A, (-)-Abz-E2B, by structurally modifying S-uniconazole, which functions as an inhibitor of CYP707A and as a gibberellin biosynthetic enzyme. However, its synthetic yield is too low for practical applications. Therefore, we designed novel CYP707A inhibitors, Abz-T compounds, that have simpler structures in which the 1,2,3-triazolyl ring of (-)-Abz-E2B has been replaced with a triple bond. They were successfully synthesised in shorter steps, resulting in greater yields than that of (-)-Abz-E2B. In the enzymatic assays, one of the Abz-T compounds, (-)-Abz-E3M, acted as a strong and selective inhibitor of CYP707A, similar to (-)-Abz-E2B. Analysis of the biological effects in Arabidopsis revealed that (-)-Abz-E3M enhanced ABA's effects more than (-)-Abz-E2B in seed germination and in the expression of ABA-responsive genes. Treatment with (-)-Abz-E3M induced stomatal closure and improved drought tolerance in Arabidopsis. Furthermore, (-)-Abz-E3M also increased the ABA response in rice and maize. Thus, (-)-Abz-E3M is a more practical and effective inhibitor of CYP707A than (-)-Abz-E2B.

Synthesis and herbicidal activity of 3-{[(hetero)aryl]methanesulfonyl}-4,5-dihydro-1,2-oxazole derivative; Discovery of the novel pre-emergence herbicide pyroxasulfone.

Thiocarbamate sulfoxides, which are the active forms of thiocarbamate herbicides, are quickly conjugated with glutathione and decomposed in soil. To achieve more potent and stable herbicidal activity, we previously developed a 5-{[(2,6-difluorophenyl)methoxy]methyl}-5-methyl derivative, which has a 4,5-dihydro-1,2-oxazole ring in place of the thiocarbamate to prevent conjugation and decomposition. Although the derivative showed pre-emergence herbicidal activity under flooded conditions, it displayed no herbicidal activity under upland conditions. In contrast, a 5-(methoxymethyl)-5-methyl derivative showed pre-emergence herbicidal activity against grass weeds under upland conditions. The aim of this study was to obtain a more potent compound with improved physicochemical properties for use as a pre-emergence upland herbicide via the structural optimization of a 3-{[(hetero)aryl]methanesulfonyl}-4,5-dihydro-1,2-oxazole as the core structure. In this way, we have developed the pre-emergence herbicide 3-{[5-(difluoromethoxy)-1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]methanesulfonyl}-5,5-dimethyl-4,5-dihydro-1,2-oxazole, which has been named "pyroxasulfone." This novel compound displayed excellent herbicidal activity against grass and broadleaf weeds under upland conditions with no phytotoxicity against crops.

The selectivity of 6-nor-ABA and 7'-nor-ABA for abscisic acid receptor subtypes.

Abscisic acid (ABA), a plant hormone, is involved in many plant development processes and environmental stress responses that are regulated by a Pyrabactin Resistant 1 (PYR)/Pyrabactin Resistant-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) receptor protein-mediated signal transduction pathway. In Arabidopsis thaliana, PYL proteins constitute a 14-member family comprising two distinct subclasses: dimeric receptors (PYR1 and PYL1-PYL3) and monomeric receptors (PYL4-PYL13). The individual contributions of PYL subclasses/subtypes with specific physiological actions are still poorly understood; consequently, the development of PYL subclass/subtype-selective agonists should be useful to reveal the different functions of these receptors. In this study, we focused on the ABA analogs 6-nor-ABA and 7'-nor-ABA, which were expected to function as monomeric receptor-selective agonists on the basis of crystal structures of PYL-ABA complexes and sequence alignments of PYL subtypes. In a protein phosphatase 2C (PP2C) assay, the agonist activities of both analogs were lower than those of ABA toward all tested PYL proteins, regardless of subclass/subtype. Nevertheless, we found that 6-nor-ABA acts as a selective agonist at the physiological level: it induced stomatal closure but did not inhibit seed germination and root growth. On the basis of observed inhibitory activity against PP2C among different PYL subtypes, this biological effect of 6-nor-ABA may be attributed to the activity of that agonist on PYL5 and/or PYL6.

Cyanamide is biosynthesized from L-canavanine in plants.

Cyanamide had long been recognized as a synthetic compound but more recently has been found as a natural product from several leguminous plants. This compound's biosynthetic pathway, as yet unelaborated, has attracted attention because of its utility in many domains, such as agriculture, chemistry, and medicine. We noticed that the distribution of L-canavanine in the plant kingdom appeared to include that of cyanamide and that the guanidino group structure in L-canavanine contained the cyanamide skeleton. Here, quantification of these compounds in Vicia species suggested that cyanamide was biosynthesized from L-canavanine. Subsequent experiments involving L-[guanidineimino-(15)N2]canavanine addition to young Vicia villosa seedlings resulted in significant incorporation of (15)N-label into cyanamide, verifying its presumed biosynthetic pathway.

Conformationally restricted 3'-modified ABA analogs for controlling ABA receptors.

The physiological functions of abscisic acid (ABA) are regulated by a signal transduction pathway involving cytosolic ABA receptors, which include 14 PYR/PYL/RCAR (PYL) proteins in Arabidopsis. The development of a PYL antagonist could be a valuable tool to improve our understanding of the roles of ABA. We previously developed 3'-hexylsulfanyl-ABA (AS6), whose S-hexyl chain blocks protein phosphatase 2C (PP2C) binding by steric hindrance. This finding not only validated our structure-based approach to the design of a PYL antagonist, but also provided a basis for the development of a more potent or subclass/subtype selective PYL antagonist. In the present study, we synthesized a conformationally restricted analog of AS6, namely propenyl-ABA with an O-butyl chain (PAO4), to improve the affinity for PYL proteins by reducing the entropic penalty for binding to the receptors. In seed germination assays, (+)-PAO4 was a slightly stronger antagonist than AS6 in Arabidopsis and a significantly stronger antagonist in lettuce. Analysis of the thermodynamic parameters associated with the formation of the Arabidopsis PYL-(+)-PAO4 complex revealed that (+)-PAO4 binds more strongly to PYL5 than AS6 owing to an entropic advantage. In PP2C assays, this enhancement effect was observed only for the monomeric PYL subclass containing PYL5, suggesting that (+)-PAO4 is more effective than AS6 in physiological events involving monomeric PYL proteins as ABA receptors.

Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

Metabolism of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Recently, we reported the conversion of bisphenol A (BPA) to 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol (hydroxy-BPA) by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition. In the present study, the metabolism of hydroxy-BPA by P. sordida YK-624 was demonstrated under non-ligninolytic condition. Under these conditions, approximately 66% of hydroxy-BPA was degraded after 7 d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of the metabolites isolated from the culture broth indicated that hydroxy-BPA was metabolized to 4-(2-(4-hydroxyphenyl)propan-2-yl)-2-methoxyphenol (methoxy-BPA) and to 4-(2-(3,4-dimethoxyphenyl)propan-2-yl)phenol (dimethoxy-BPA) by sequential methylation events. These metabolites showed reduced estrogenic activity compared to BPA. These results suggested that the hydroxy BPA is methylated to two low toxic-methylation metabolites.

Brassinolide-2,3-acetonide: a brassinolide-induced rice lamina joint inclination antagonist.

A novel chemical tool compound that is an antagonist of brassinolide (BL, 1)-induced rice lamina joint inclination was developed. Although 2-O-, 3-O-, 22-O-, or 23-O-methylation of BL causes a critical decrease in biological activity,(5) a crystal structure of the extracellular leucine-rich repeat (LRR) domain of BRASSINOSTEROID-INSENSITIVE I (BRI1) bound to BL(3,4) indicates that the loss of activity of the O-methylated BL may result from not only the low affinity to BRI1, but also from blocking the interaction with another BR signaling factor, a partner protein of BRI1 (e.g., BRI1-ASSOCIATED KINASE 1, BAK1). On the basis of this hypothesis we synthesized the BL 2,3-acetonide 2, the 22,23-acetonide 3, and the 2,3:22,23-diacetonide 4 to assess the possibility of 2-O- and 3-O- or/and 22-O- and 23-O-alkylated BL as an antagonist in BR signaling evoked by exogenously applied BL. The 2,3-acetonide 2 more strongly inhibited the lamina inclination caused by BL relative to the 22,23-acetonide 3, whereas the diacetonide 4 had no effect most likely due to its increased hydrophobicity. This suggested that the 2,3-hydroxyl groups of BL play a more significant role in the interaction with a BRI1 partner protein rather than BRI1 itself in rice lamina joint inclination. Taken together it was demonstrated that BL, the most potent agonist of BRI1, is transformed into an antagonist by functionalization of the 2,3-dihydroxyl groups as the acetonide. This finding opens the door to the potential development of a chemical tool that modulates protein-protein interactions in the BR signaling pathway to dissect the BR-dependent processes.

Influence of exogenously applied abscisic acid on carotenoid content and water uptake in flowers of the tea plant (Camellia sinensis).

BACKGROUND: Carotenoids are a major class of plant pigments and fulfill many functions in different organisms that either produce or consume them. Although the color of the stamina of tea (Camellia sinensis) flowers is clearly due to the presence of carotenoids, the carotenoid profile and content remain to be discovered. RESULTS: We investigated the carotenoid profile of tea flowers and determined changes in concentrations over the floral development. The flowers contained oxygenated xanthophylls such as neoxanthin, lutein and zeaxanthin, as well as the hydrocarbons ß-carotene and α-carotene. Flowers of the tea plant contain to vegetables comparable amounts of carotenoids. The content of 9'-cis-epoxycarotenoids, which serve as abscisic acid precursors, as well as changes in concentration of abscisic acid were studied. The concentrations of carotenoids decreased whereas the abscisic acid content increased over the floral development. Exogenously applied S-abscisic acid affected water uptake, flower opening and carotenoid accumulation. CONCLUSION: In summary, this paper reports, for the first time, the carotenoid profile and content of tea flowers. The study revealed that carotenoids in tea flowers are an interesting target in respect of possible applications of tea flower extracts as well as biological functions of abscisic acid during floral development.

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24.

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8'-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18 h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2B, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds.

Electrolytic reduction of abscisic acid methyl ester and its free acid.

Abscisic acid (ABA, 1), a plant hormone, has electrophilicity derived almost entirely from the side-chain, 3-methylpenta-2,4-dienoic acid. The electrochemical property of ABA was investigated by analysis of its cathodic reaction. ABA methyl ester (1-Me) was reduced at a peak potential of -1.6 V to give a unique and unstable bicyclic compound (5-Me) as a major product at pH 3 and 7. This finding showed that an electron was absorbed in the conjugated dienecarboxyl group, and that C-5 with a high electron density attacked C-2' through an intramolecular nucleophilic addition. At pH 10, in addition to 5-Me, a compound 4-Me was formed by isomerization of 5-Me under alkaline conditions. For a cathodic reaction of ABA at pH 3 and 7, compound 5 was a major product as well as in the case of ABA methyl ester. However, at pH 10, a dimer (6) with an epoxy group, 1'-deoxy-ABA (7) and other compounds were formed instead of compounds 4 and 5. Compounds 4 and 5 were biologically inactive, suggesting the importance of the electrophilic side-chain of ABA for biological activity.

Abscinazole-E2B, a practical and selective inhibitor of ABA 8'-hydroxylase CYP707A.

We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated logP value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 µM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 µM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 µM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.