Tetsuo Tokiwano

Solanapyrone synthase, a possible Diels-Alderase and iterative type I polyketide synthase encoded in a biosynthetic gene cluster from Alternaria solani.

The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes—sol1–6—coding for a polyketide synthase, an O‐methyltransferase, a dehydrogenase, a transcription factor, a flavin‐dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels–Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin‐dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (−)‐solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels–Alder enzyme.

Epoxide Hydrolase Lsd19 for Polyether Formation in the Biosynthesis of Lasalocid A: Direct Experimental Evidence on Polyene-Polyepoxide Hypothesis in Polyether Biosynthesis

Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.

Biosynthetic Gene-Based Secondary Metabolite Screening: A New Diterpene, Methyl Phomopsenonate, from the Fungus Phomopsis amygdali

The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.

Identification of a Gene Cluster of Polyether Antibiotic Lasalocid from Streptomyces lasaliensis

Elucidation of enzymatic polyether formation is a long-standing controversial issue in organic chemistry. To address this intriguing issue, identifying the actual substrate for epoxidation and sequential cyclization is essential. We selected the representative polyether ionophore, lasalocid, which has been proposed to undergo no modification at the late stage of biosynthesis. Cloning and a sequence analysis revealed seven polyketide synthase (PKS) genes, epoxidase and epoxide hydrolase genes for sequential ether formation, and several putative genes for supplying ethylmalonyl-CoA. Based on bioinformatic data, we propose the lasalocid biosynthetic pathway which involves characteristic aromatic ring formation and sequential cyclic ether formation. The finding of a thioesterase domain at the C-terminal of the seventh PKS indicates that intriguing oxidative cascade cyclization would occur after cleavage of the polyketide intermediate from PKS. Based on this observation, we have recently reported the enzymatic transformation of a bisepoxide intermediate to lasalocid with the recombinant epoxide hydrolase, Lsd19.

LA(OTF)3-CATALYZED 7-ENDO AND 8-ENDO SELECTIVE CYCLIZATIONS OF HYDROXY EPOXIDES

Abstract Endo selective intramolecular cyclization has been achieved in 5, 6-epoxy-5-methoxymethyl-1-heptanol and 6, 7-epoxy-6-methoxymethyl-3-octen-1-ol systems by the chelation of La(OTf) 3 between the oxygen atoms of the epoxide and methoxymethyl groups, affording 3-hydroxyoxepane and 3-hydroxy-5-oxocene derivatives, respectively.

La(otf)3-catalyzed 6-endo epoxide opening of 4, 5-epoxy-4-methoxymethyl-1-hexanols

Abstract The selective 6-endo epoxide ring opening of cis- and trans-4, 5-epoxy-4-melhoxymethyl-1-hexanols has been achieved by the chelation of La(OTf)3·nH2O between the oxygens of the epoxide and the methoxymethyl groups to afford 3-hydroxytetrahydropyran derivatives stereospecifically.

CHEMICAL REALIZATION OF THE BIOGENETIC PATHWAYS PROPOSED FOR THE FUSED-POLYCYCLIC ETHERS OF MARINE ORIGINS

The biomimetic constructions of fused polycyclic ethers of marine origins according to the proposals for their biogenetic pathways starting from trans-polyepoxide compounds could be realized chemically as the model experiments in two ways : one is regarded epoxy groups as electrophiles and other is those as nucleophiles.

Intriguing Substrate Tolerance of Epoxide Hydrolase Lsd19 Involved in Biosynthesis of the Ionophore Antibiotic Lasalocid A

Recently, we reported that the epoxide hydrolase Lsd19, the first enzyme shown to catalyze epoxide-opening cascades, can catalyze the conversion of a putative bisepoxide intermediate to polyether antibiotic lasalocid, which involves energetically disfavored 6-endo-tet cyclization of the epoxy alcohol. Here, we examined the substrate tolerance of Lsd19. Lsd19 accepts various substrate analogues differing in the left segment of lasalocid and epoxide stereochemistry to afford either THF-THP or THF-THF products with excellent regioselectivity.

A Lichen Substance as an Antiproliferative Compound against HL-60 Human Leukemia Cells: 16-O-Acetyl-leucotylic Acid Isolated from Myelochroa aurulenta

The lichen substance, 16-O-acetyl-leucotylic acid (1), was isolated from an acetone extract of Myelochroa aurulenta and found to exhibit antiproliferative activity against HL-60 human leukemia cells. This is the first report on its anti-leukemia activity (EC50=21 μM) which is greater than that of leucotylic acid (2) and the structurally related anti-tumor agent, betulinic acid (4).

Biosynthesis of phomactins: common intermediate phomactatriene and taxadieneElectronic supplementary information (ESI) available: 1H and 13C NMR spectra for labelled and unlabelled 1.See http://www.rsc.org/suppdata/cc/b4/b401377h/

The stereochemical course of GGDP cyclization in the biosynthesis of phomactins is proposed by the stereochemistry of a cyclization product, phomacta-1(14),3,7-triene, isolated from Phomasp. and the results of incorporation experiments with [1-13C]- and [1,2-13C2]acetates.