Total Synthesis of Bipenicilisorin and Assignment of the Absolute Configuration.

The first total synthesis of bipenicilisorin (1) isolated from Penicillium chrysogenum SCSIO 41001 via its monomer natural product, penicilisorin (2), was achieved. Penicilisorin was synthesized in four steps from a o-bromobenzaldehyde derivative via the Pd-catalyzed one-pot fluorocarbonylation/lactonization/ß-elimination cascade reaction. Iodination of penicilisorin gave 7-iodopenicilisorin which was dimerized by Pd-catalyzed homodimerization to provide (±)-bipenicilisorin. The unknown absolute configuration of naturally occurring (+)-bipenicilisorin was examined by optical resolution of the (±)-synthetic bipenicilisorin and a comparison of experimental and theoretical electronic circular dichroism (ECD) spectra. These results support the absolute configuration of the natural product to be Sa. A cytotoxic activity test of (+)-and (-)-bipenicilisorin using A549 cells revealed that (+)-1 has a lower IC50 value than (-)-1.

Substrate-Dependent Alteration in the C- and O-Prenylation Specificities of Cannabis Prenyltransferase.

CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.

Catalytic Potential of Cannabis Prenyltransferase to Expand Cannabinoid Scaffold Diversity.

Biologically active cannabinoids are derived from cannabigerolic acid (CBGA), which is biosynthesized by aromatic prenyltransferase CsPT4. We exploit the catalytic versatility of CsPT4 to synthesize various CBGA analogues, including a geranylated bibenzyl acid, the precursor to bibenzyl cannabinoids of liverwort origin. The synthesized natural and new-to-nature cannabinoids exhibit potent cytotoxicity in human pancreatic cancer cells. CsPT4 can artificially extend the cannabinoid biosynthetic diversity with novel and improved biological activities.

A [4Fe-4S] cluster resides at the active center of phosphomevalonate dehydratase, a key enzyme in the archaeal modified mevalonate pathway.

The recent discovery of the archaeal modified mevalonate pathway revealed that the fundamental units for isoprenoid biosynthesis (isopentenyl diphosphate and dimethylallyl diphosphate) are biosynthesized via a specific intermediate, trans-anhydromevalonate phosphate. In this biosynthetic pathway, which is unique to archaea, the formation of trans-anhydromevalonate phosphate from (R)-mevalonate 5-phosphate is catalyzed by a key enzyme, phosphomevalonate dehydratase. This archaea-specific enzyme belongs to the aconitase X family within the aconitase superfamily, along with bacterial homologs involved in hydroxyproline metabolism. Although an iron-sulfur cluster is thought to exist in phosphomevalonate dehydratase and is believed to be responsible for the catalytic mechanism of the enzyme, the structure and role of this cluster have not been well characterized. Here, we reconstructed the iron-sulfur cluster of phosphomevalonate dehydratase from the hyperthermophilic archaeon Aeropyrum pernix to perform biochemical characterization and kinetic analysis of the enzyme. Electron paramagnetic resonance, iron quantification, and mutagenic studies of the enzyme demonstrated that three conserved cysteine residues coordinate a [4Fe-4S] cluster-as is typical in aconitase superfamily hydratases/dehydratases, in contrast to bacterial aconitase X-family enzymes, which have been reported to harbor a [2Fe-2S] cluster.

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.

Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for o-Dialkylbenzene Biosynthesis.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

Catalytic Asymmetric Hydrogenation of Dehydroamino Acid Esters with Biscarbamate Protection and Its Application to the Synthesis of xCT Inhibitors.

Catalytic asymmetric hydrogenation of dehydroamino acid esters with biscarbamate protection was examined for the first time to prepare optically active amino acids. The new method was successfully applied to the synthesis of new cystine-glutamate exchanger inhibitors.

An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid.

Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata.

BACKGROUND: Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. METHODS: The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. RESULTS: Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. CONCLUSION: We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Abstract Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.(AU)

Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni.

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.9 that were expected to be novel based on a peptide database search. Purification of the crude venom by HPLC gave two peptide fractions, P-1 and P-2. The amino acid sequence of P-1 (GRILSFIKAGLAEHL-NH2) and P-2 (ILGIITSLLKSL-NH2) were determined by ESI-MS/MS, automated Edman degradation, and amino acid analysis. According to the high sequence homology with those of mastoparan and protonectin, P-1 and P-2 were labeled Eumenine mastoparan-OD and Orancis-Protonectin, respectively. Orancis-Protonectin is the first example of a protonectin analog isolated from the venom of a solitary wasp. The hemolytic activities of these new peptides were more potent than that of mastoparan.

A quinone isolated from the nest of Vespa simillima and its growth-inhibitory effect on rat liver cancer cells.

A biologically active quinone that exhibits a growth-inhibitory effect on rat liver cancer cells was isolated from the nest of a social wasp, Vespa simillima (Vespinae, Vespidae). The structure was unambiguously determined by NMR and synthetic experiments and identified as being 7,8-seco-para-ferruginone. The synthetic quinone showed potent growth-inhibitory effects on rat hepatoma cells (IC50 value=1.9 microM). Morphological analyses of dRLh84 cells using the synthetic compound were tested. Chromatin coagulation was induced by the synthetic compound at 31.6 muM.

Novel biologically active peptides from the venom of Polistes rothneyi iwatai.

Four novel peptides, polistes-mastoparan-R1, 2, 3, and polistes-protonectin, were isolated from the venom of a paper wasp, Polistes rothneyi iwatai. MALDI-TOF MS analysis of a small amount of the crude venom gave six molecular-related ion peaks. Among them, m/z 1565 was expected to be a novel peptide. Purification of the crude venom by HPLC gave two known kinins, Thr6-bradykinin and Ala-Arg-Thr6-bradykinin, and four novel peptides named polistes-mastoparan-R1, 2, and 3, and polistes-protonectin. Polistes-mastoparan-R1, 2, and 3 (Pm-R) were tetradecapeptides that possess high sequence homology with that of mastoparan. The sequence of polistes-protonectin was similar to that of protonectin isolated from a Brazilian paper wasp. Histamine-releasing activities of Pm-R1, 2, and 3 were more potent than that of mastoparan. Polistes-protonectin exhibited the most potent hemolytic activity in comparison with the four novel peptides and mastoparan.