Insights into stereoselective ring formation in canonical strigolactone: Identification of a dirigent domain-containing enzyme catalyzing orobanchol synthesis.

Strigolactones (SLs) are plant apocarotenoids with diverse roles and structures. Canonical SLs, widespread and characterized by structural variations in their tricyclic lactone (ABC-ring), are classified into two types based on C-ring configurations. The steric C-ring configuration emerges during the BC-ring closure, downstream of the biosynthetic intermediate, carlactonoic acid (CLA). Most plants produce either type of canonical SLs stereoselectively, e.g., tomato (Solanum lycopersicum) yields orobanchol with an α-oriented C-ring. The mechanisms driving SL structural diversification are partially understood, with limited insight into functional implications. Furthermore, the exact molecular mechanism for the stereoselective BC-ring closure reaction is yet to be known. We identified an enzyme, the stereoselective BC-ring-forming factor (SRF), from the dirigent protein (DIR) family, specifically the DIR-f subfamily, whose biochemical function had not been characterized, making it a key enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We first confirm the precise catalytic function of the tomato cytochrome P450 SlCYP722C, previously shown to be involved in orobanchol biosynthesis [T. Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. We then show that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid, forming orobanchol. Our methodology combines experimental and computational techniques, including SRF structure prediction and conducting molecular dynamics simulations, suggesting a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for the stereoselective BC-ring formation in orobanchol. This study sheds light on the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

Absolute Configuration of 4-Chloroisoleucine in Cyclolithistide A: An Antifungal Peptide Lactone Discovered in Marine Lithistid Sponges.

Cyclolithistide A is a peptide lactone isolated from marine lithistid sponges. Its entire structure, including absolute configurations, has been reported except the relative and absolute configurations of its characteristic residue, 4-chloroisoleucine (4-CIle). We synthesized four isomers of 4-CIle from furfural-derived N-Boc imine and propionaldehyde. Analysis of the acid hydrolysate of cyclolithistide A and the synthetic samples of 4-CIle after derivatization with l- and d-FDAA permitted us to propose the absolute configuration of the 4-chloroisoleucine residue in cyclolithistide A as 2S,3R,4R.

2-oxoglutarate-dependent dioxygenases and BAHD acyltransferases drive the structural diversification of orobanchol in Fabaceae plants.

Strigolactones (SLs), a class of plant apocarotenoids, serve dual roles as rhizosphere-signaling molecules and plant hormones. Orobanchol, a major naturally occurring SL, along with its various derivatives, has been detected in the root exudates of plants of the Fabaceae family. Medicaol, fabacyl acetate, and orobanchyl acetate were identified in the root exudates of barrel medic (Medicago truncatula), pea (Pisum sativum), and cowpea (Vigna unguiculata), respectively. Although the biosynthetic pathway leading to orobanchol production has been elucidated, the biosynthetic pathways of the orobanchol derivatives have not yet been fully elucidated. Here, we report the identification of 2-oxoglutarate-dependent dioxygenases (DOXs) and BAHD acyltransferases responsible for converting orobanchol to these derivatives in Fabaceae plants. First, the metabolic pathways downstream of orobanchol were analyzed using substrate feeding experiments. Prohexadione, an inhibitor of DOX inhibits the conversion of orobanchol to medicaol in barrel medic. The DOX inhibitor also reduced the formation of fabacyl acetate and fabacol, a precursor of fabacyl acetate, in pea. Subsequently, we utilized a dataset based on comparative transcriptome analysis to select a candidate gene encoding DOX for medicaol synthase in barrel medic. Recombinant proteins of the gene converted orobanchol to medicaol. The candidate genes encoding DOX and BAHD acyltransferase for fabacol synthase and fabacol acetyltransferase, respectively, were selected by co-expression analysis in pea. The recombinant proteins of the candidate genes converted orobanchol to fabacol and acetylated fabacol. Furthermore, fabacol acetyltransferase and its homolog in cowpea acetylated orobanchol. The kinetics and substrate specificity analyses revealed high affinity and strict recognition of the substrates of the identified enzymes. These findings shed light on the molecular mechanisms underlying the structural diversity of SLs.

Syntheses of (+)-costic acid and structurally related eudesmane sesquiterpenoids and their biological evaluations as acaricidal agents against Varroa destructor.

Synthesis of (+)-costic acid, isolated from Dittrichia viscosa (L.) W. Greuter as a natural acaricidal sesquiterpenoid, was achieved in 16 steps from (R)-carvone with an overall yield of 4.8%, involving the radical cyclization of selenoester to construct a decalone framework as the key step. Other structurally related natural products, (+)-costal, (+)-costol, and (+)-ß-selinene, were also synthesized. The acaricidal activities of these four natural products and some synthetic intermediates were also evaluated against Varroa destructor. Among them, (+)-costal especially exhibited potent acaricidal activity.

A step-economical divergent approach to isoflavenes based on Suzuki-Miyaura cross coupling of a 3-boryl-2H-chromene with aryl bromides: application to total synthesis of isoflavonoid natural products.

We present a step-economical divergent synthetic approach for isoflavene derivatives using the Suzuki-Miyaura cross coupling of a 3-boryl-2H-chromene and three aryl bromides. 3-Boryl-2H-chromene, which is not a well-explored species, was prepared via Miyaura-Ishiyama borylation of a 3-chloro-2H-chromene obtained through a Claisen rearrangement cyclization cascade reaction. Further conversion of the cross-coupling products, three isoflavene derivatives, afforded three isoflavonoid natural products with one or two additional reaction steps.

Overview of Kenji Mori's pheromone synthesis series.

It was the late Professor Kenji Mori, the giant of pheromone synthesis and pioneer of pheromone stereochemistry, who laid the foundation for the practical application of insect pheromones, which play an important role in Integrated Pest Management, one of the key concepts of agriculture in the 21st century. Therefore, it would be meaningful to retrace his achievements at this time, three and a half years after his death. In this review, we would like to introduce some of his notable synthetic studies from his Pheromone Synthesis Series and reconfirm his contributions to the development of pheromone chemistry and their impacts on natural science.

Corrigendum: Identification of novel canonical strigolactones produced by tomato.

[This corrects the article DOI: 10.3389/fpls.2022.1064378.].

Mechanisms of Sugar Aminotransferase-like Enzymes to Synthesize Stereoisomers of Non-proteinogenic Amino Acids in Natural Product Biosynthesis.

(2,6)-Diamino-(5,7)-dihydroxyheptanoic acid (DADH), a non-proteinogenic amino acid, is converted to 1-azabicyclo[3.1.0]hexane ring-containing amino acids that are subsequently incorporated into ficellomycin and vazabitide A. The present study revealed that the sugar aminotransferase-like enzymes Fic25 and Vzb9, with a high amino acid sequence identity (56%) to each other, synthesized stereoisomers of DADH with (6S) and (6R) configurations, respectively. The crystal structure of the Fic25 complex with a PLP-(6S)-N2-acetyl-DADH adduct indicated that Asn45 and Gln197 (Asn205 and Ala53 in Vzb9) were located at positions that affected the stereochemistry of DADH being synthesized. A modeling study suggested that amino acid substitutions between Fic25 and Vzb9 allowed the enzymes to bind to the substrate with almost 180° rotation in the C5-C7 portions of the DADH molecules, accompanied by a concomitant shift in their C1-C4 portions. In support of this result, the replacement of two corresponding residues in Fic25 and Vzb9 increased (6R) and (6S) stereoselectivities, respectively. The different stereochemistry at C6 of DADH resulted in a different stereochemistry/orientation of the aziridine portion of the 1-azabicyclo[3.1.0]hexane ring, which plays a crucial role in biological activity, between ficellomycin and vazabitide A. A phylogenic analysis suggested that Fic25 and Vzb9 evolved from sugar aminotransferases to produce unusual building blocks for expanding the structural diversity of secondary metabolites.

New synthesis of (±)-karanone, an important aroma compound in agarwood.

A concise synthesis of (±)-karanone, an important aroma compound of agarwood, was achieved from a commercially available 3-methylcyclohex-2-enol in 3.5% yield in 11 steps. The two contiguous stereocenters at C4 and C5 were constructed via Ireland-Claisen rearrangement. The allylic oxidation at C8 was successfully performed with the mixture of tert-butyl hydroperoxide (TBHP) and CuI. A precursor of ring-closing metathesis to construct a bicyclic dienone was efficiently synthesized from iodoenone by 1,4-addition and nucleophilic substitution of the vinyl group in one pot.

Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Identification of 6-epi-heliolactone as a biosynthetic precursor of avenaol in Avena strigosa.

Strigolactones (SLs), which are known as rhizosphere signaling molecules and plant hormones regulating shoot architecture, are classified into 2 distinct groups, canonical and noncanonical SLs, based on their structures. Avenaol, a noncanonical SL found in the root exudates of black oat (Avena strigosa), has a characteristic bicyclo[4.1.0]heptane skeleton. Elucidating the biosynthetic mechanism of this peculiar structure is a challenge for further understanding of the structural diversification of noncanonical SLs. In this study, a novel noncanonical SL, 6-epi-heliolactone in black oat root exudates was identified. Feeding experiments showed that 6-epi-heliolactone was a biosynthetic intermediate between methyl carlactonoate and avenaol. Inhibitor experiments proposed the involvement of 2-oxoglutarate-dependent dioxygenase in converting 6-epi-heliolactone to avenaol. These results provide new insights into the stereochemistry diversity of noncanonical SLs and a basis to explore the biosynthetic pathway causing avenaol.

Synthesis and biological evaluation of cajaninstilbene acid and amorfrutins A-D as cytotoxic agents against human pancreatic carcinoma PANC-1 cells.

A concise synthesis of cajaninstilbene acid was achieved in 7 steps from (E)-3,5-dimethoxystilbene in 8.6% overall yield via the Claisen rearrangement of an aryl reverse-prenyl ether as the key step. Cytotoxic activities against human pancreatic carcinoma PANC-1 cells of cajaninstilbene acid and amorfrutins A-D were also evaluated.

Conversion of methyl carlactonoate to heliolactone in sunflower.

Heliolactone is a non-canonical strigolactone isolated from sunflower root exudates. We have previously demonstrated that exogenously administered carlactonoic acid (CLA) was converted to heliolactone in sunflower. The conversion of CLA to heliolactone requires the methyl esterification of the carboxylic acid at C-19. Also, the CLA conversion to its methyl ester, methyl carlactonoate (MeCLA), was demonstrated by feeding experiment in sunflower. However, the involvement of MeCLA in heliolactone biosynthesis remains unclear. We synthesised MeCLA in its racemic form and resolved it into its enantiomers. Feeding experiments revealed that (11R)-MeCLA was exclusively converted to heliolactone in sunflower. This result is an evidence that (11R)-MeCLA is the biosynthetic precursor of heliolactone. Further conversion of heliolactone to an unidentified metabolite with a molecular mass larger than heliolactone by 16 Da was confirmed. The conversion was inhibited by a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450-dependent monooxygenation.

Enantioselective synthesis of 6-methyloctanal and 8-methyldecanal, the characteristic aroma components in yuzu Citrus junos, and the analysis of their enantiomeric compositions in yuzu essential oil.

6-Methyloctanal and 8-methyldecanal are the characteristic aroma components of yuzu Citrus junos. However, their absolute configurations and enantiomeric compositions in yuzu essential oil have not been analyzed. A concise enantioselective synthesis of both aldehydes was successfully carried out to determine their absolute configurations and enantiomeric compositions. Both aldehydes in yuzu essential oil were found to be (S)-form with high enantiomeric excess.

Identification of novel canonical strigolactones produced by tomato.

Canonical strigolactones (SLs), such as orobanchol, consist of a tricyclic lactone ring (ABC-ring) connected to a methylbutenolide (D-ring). Tomato plants have been reported to produce not only orobanchol but also various canonical SLs related to the orobanchol structure, including orobanchyl acetate, 7-hydroxyorobanchol isomers, 7-oxoorobanchol, and solanacol. In addition to these, structurally unidentified SL-like compounds known as didehydroorobanchol isomers (DDHs), whose molecular mass is 2 Da smaller than that of orobanchol, have been found. Although the SL biosynthetic pathway in tomato is partially characterized, structural elucidation of DDHs is required for a better understanding of the entire biosynthetic pathway. In this study, three novel canonical SLs with the same molecular mass as DDHs were identified in tomato root exudates. The first was 6,7-didehydroorobanchol, while the other two were not in the DDH category. These two SLs were designated phelipanchol and epiphelipanchol because they induced the germination of Phelipanche ramosa, a noxious root parasitic weed of tomato. We also proposed a putative biosynthetic pathway incorporating these novel SLs from orobanchol to solanacol.

Specific methylation of (11R)-carlactonoic acid by an Arabidopsis SABATH methyltransferase.

MAIN CONCLUSION: An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 µM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.

Homophymamide A, Heterodetic Cyclic Tetrapeptide from a Homophymia sp. Marine Sponge: A Cautionary Note on Configurational Assignment of Peptides That Contain a Ureido Linkage.

A previously unreported heterodetic cyclic peptide, homophymamide A (1), was isolated from a Homophymia sp. marine sponge. The structure of homophymamide A was determined to be a lower homologue of anabaenopeptins by spectroscopic analysis, chemical degradation, and chemical synthesis. Analysis of the acidic hydrolysate showed that the racemization of Lys took place, leading us to pose a cautionary note on the configurational assignment of peptides that contain a ureido bond.

JBIR-155, a Specific Class D ß-Lactamase Inhibitor of Microbial Origin.

We discovered JBIR-155 as a novel specific class D ß-lactamase inhibitor from Streptomyces polymachus SoB100815Hv02. JBIR-155 consists of a 6-oxabicyclo[3.2.0]heptan-7-one skeleton and a long unsaturated alkyl chain moiety of which absolute configuration was determined by spectroscopic data, modified Mosher's method, and analyses of the relative configuration of chemically modified derivative. JBIR-155 specifically exhibited inhibitory activity against the class D ß-lactamase, with an IC50 value of 0.36 µM.

Identification and characterization of sorgomol synthase in sorghum strigolactone biosynthesis.

Strigolactones (SLs), first identified as germination stimulants for root parasitic weeds, act as endogenous phytohormones regulating shoot branching and as root-derived signal molecules mediating symbiotic communications in the rhizosphere. Canonical SLs typically have an ABCD ring system and can be classified into orobanchol- and strigol-type based on the C-ring stereochemistry. Their simplest structures are 4-deoxyorobanchol (4DO) and 5-deoxystrigol (5DS), respectively. Diverse canonical SLs are chemically modified with one or more hydroxy or acetoxy groups introduced into the A- and/or B-ring of these simplest structures, but the biochemical mechanisms behind this structural diversity remain largely unexplored. Sorgomol in sorghum (Sorghum bicolor [L.] Moench) is a strigol-type SL with a hydroxy group at C-9 of 5DS. In this study, we characterized sorgomol synthase. Microsomal fractions prepared from a high-sorgomol-producing cultivar of sorghum, Sudax, were shown to convert 5DS to sorgomol. A comparative transcriptome analysis identified SbCYP728B subfamily as candidate genes encoding sorgomol synthase. Recombinant SbCYP728B35 catalyzed the conversion of 5DS to sorgomol in vitro. Substrate specificity revealed that the C-8bS configuration in the C-ring of 5DS stereoisomers was essential for this reaction. The overexpression of SbCYP728B35 in Lotus japonicus hairy roots, which produce 5DS as an endogenous SL, also resulted in the conversion of 5DS to sorgomol. Furthermore, SbCYP728B35 expression was not detected in nonsorgomol-producing cultivar, Abu70, suggesting that this gene is responsible for sorgomol production in sorghum. Identification of the mechanism modifying parental 5DS of strigol-type SLs provides insights on how plants biosynthesize diverse SLs.