Robert M. Williams

Isolation, Structure Elucidation, and Biomimetic Total Synthesis of Versicolamide B, and the Isolation of Antipodal (−)-Stephacidin A and (+)-Notoamide B from Aspergillus versicolor NRRL 35600†

Prenylated indole alkaloids containing, the bicyclo[2.2.2]diazaoctane ring system as a structural core, now number more than thirty-eight family members. These natural substances, produced by various genera of fungi, in particular Aspergillus sp. and Penicillium spp., among others, exhibit a range of interesting structural and stereochemical features. Significantly, a myriad of biological activities are displayed by members of this family including insecticidal, anti-tumor, anthelmintic, calmodulin inhibitory, and anti-bacterial activities. Structurally, these substances arise from the oxidative condensation of one or two isoprene units, tryptophan and another cyclic amino acid residue, such as proline, β-methylproline or pipecolic acid. With respect to the relative stereochemistry within the core bicyclo[2.2.2]diazaoctane ring system, all of the known members of the paraherquamides (e.g., 1, 2)/stephacidins (e.g., 3, 4)/asperparalines and notoamides (e.g., 5, 6) have been shown to possess the syn-stereochemistry, while only the brevianamides (9, 10) have been shown to possess the anti-relative configuration (Schemes 1 and ​and2).2). The syn-/anti-relationship refers to the relative stereochemistry between the C-19 stereogenic center (sclerotiamide numbering) and the cyclic amino acid residue (proline, β-methylproline, or pipecolic acid; Scheme 2). This reveals that in the oxidative cyclization process(es) to construct this core ring system biosynthetically, both faces of the isoprene-derived dienophile participate in the ring-forming process. However, until now, this stereochemical divergence was cleanly separated between the brevianamides and all other members of this growing family of natural products. Herein, we describe the isolation, structure elucidation, and confirmatory biomimetic total synthesis of the first member of the paraherquamide-stephacidin family to possess the rare anti-relative stereochemistry within the bicyclo[2.2.2]diazaoctane ring system. We propose the new name versicolamide B for this natural product (8), a minor metabolite of Aspergillus versicolor NRRL 35600, Based on CD spectra, we have assigned the absolute configuration to this compound, and have concluded that it possesses the ent-configuration with respect to the bicyclo[2.2.2]diazaoctane core. Surprisingly and as striking, we have also isolated (−)-stephacidin A and (+)-notoamide B from Aspergillus versicolor NRRL 35600 and conclude that these substances are produced as the corresponding antipodes to the structures that have been previously described for these natural products. The provocative biogenetic implications of these stereochemical findings are discussed herein.

Taxol biosynthesis: Taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10β-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5α-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10β-hydroxylase) as a taxane 13α-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5a-o1: the first oxygenation step in taxol biosynthesis

BACKGROUND The structural complexity of taxol dictates continued reliance on biological production methods, which may be improved by a detailed understanding of taxol biosynthesis, especially the rate-limiting steps. The biosynthesis of taxol involves the cyclization of the common isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5), 11(2)-diene followed by extensive, largely oxidative, modification of this diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis. RESULTS Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadiene. We confirmed the structure of the reaction product (taxa-4(20), 11(12)-dien-5alpha-ol) by synthesizing this compound. The responsible biological catalyst was characterized as a cytochrome P450 (heme thiolate protein). In vivo studies confirmed that taxadienol is a biosynthetic intermediate and indicated that the hydroxylation step that produces this product is slow relative to subsequent metabolic transformations. CONCLUSIONS The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic possibilities for subsequent elaboration of the oxetane moiety of taxol. The reaction is catalyzed by a cytochrome P450, suggesting that the seven remaining oxygenation steps in taxol biosynthesis may involve similar catalysts. Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxol biosynthesis by isolating and manipulating the gene for the taxadiene-5-hydroxylase that catalyzes this reaction.

Biosynthesis of Prenylated Alkaloids Derived from Tryptophan

The biosynthesis of prenylated indole alkaloids and related natural substances derived from tryptophan is reviewed. The families of compounds covered in this review include the brevianamides, austamides, paraherquamides, marcfortine, roquefortine, aszonalenin, echinulin, verruculogen, the fumitremorgins, a-cyclopiazonic acid, and the ergot alkaloids. Although other families of naturally occurring prenylated indole alkaloids exist, such as the iridoids, this review is intended to examine the biosynthesis of the groups selected based on their structural and biogenetic similarities. In addition, the biosynthesis of the families selected for this chapter have not, to the best of our knowledge, been previously reviewed.

Total synthesis and biological mode of action of largazole: A potent class I histone deacetylase inhibitor

The efficient total synthesis of the recently described natural substance largazole (1) and its active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded in 37% overall yield. It is demonstrated that largazole is a pro-drug that is activated by removal of the octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite 2, which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and 2 have been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell lines, and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference is attributed to differences in the cell permeability of the two substances.

Enantiomeric Natural Products: Occurrence and Biogenesis

In nature, chiral natural products are usually produced in optically pure form-however, occasionally both enantiomers are formed. These enantiomeric natural products can arise from a single species or from different genera and/or species. Extensive research has been carried out over the years in an attempt to understand the biogenesis of naturally occurring enantiomers; however, many fascinating puzzles and stereochemical anomalies still remain.

Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process. On the basis of structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium, we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anticancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogues through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

Asymmetric total syntheses of (+)- and (−)-versicolamide B and biosynthetic implications

The Diels-Alder reaction is one of the most well-studied, synthetically useful organic transformations. While a significant number of naturally occurring substances are postulated to arise by biosynthetic Diels-Alder reactions, rigorous confirmation of a mechanistically distinct natural Diels-Alderase enzyme remains elusive. Within this context, several related fungi within the Aspergillus genus produce a number of metabolites of opposite absolute configuration including (+)- or (-)-versicolamide B. These alkaloids are hypothesized to arise via biosynthetic Diels-Alder reactions implying that each Aspergillus species possesses enantiomerically distinct Diels-Alderases. Herein, experimental validation of these biosynthetic proposals via deployment of the IMDA reaction as a key step in the asymmetric total syntheses of (+)- and (-)-versicolamide B is described. Laboratory validation of the proposed biosynthetic Diels-Alder construction, coupled with the secondary metabolite profile of the producing fungi, reveals that each Aspergillus species has evolved enantiomerically distinct indole oxidases, as well as enantiomerically distinct Diels-Alderases.

Synthesis and Conformation-Activity Relationships of the Peptide Isosteres of FK228 and Largazole

The peptide isosteres (10 and 11) of the naturally occurring and potent histone deacetylase (HDAC) inhibitors FK228 and largazole have been synthesized and evaluated side-by-side with FK228, largazole, and SAHA for inhibition of the class I HDACs 1, 2, 3, and 6.

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.