Oxygenated Theonellastrols: Interpretation of Unusual Chemical Behaviors Using Quantum Mechanical Calculations and Stereochemical Reassignment of 7α-Hydroxytheonellasterol.

A total of eight new oxygenated 4-exo-methylene sterols, 1-8, together with one artifact 9 and six known sterols 11-16, were isolated from the marine sponge Theonella swinhoei collected from the Bohol province in Philippines. Structures of sterols 1-8 were determined from 1D and 2D NMR data. Among the sterols, 8α-hydroxytheonellasterol (4) spontaneously underwent an allylic 1,3-hydroxyl shift to produce 15α-hydroxytheonellasterol (9) as an artifact; this was rationalized by quantum mechanical calculations of the transition state. In addition, the 1,2-epoxy alcohol subunit of 8α-hydroxy-14,15-ß-epoxytheonellasterol (5) was assigned using the Gauge-Independent Atomic Orbital (GIAO) NMR chemical shift calculations and subsequent DP4+ analysis. Finally, comparison of the 13C chemical shifts of isolated 7α-hydroxytheonellasterol (6) with the reported values revealed significant discrepancies at C-6, C-7, C-8, and C-14, leading to reassignment of the C-7 stereochemistry in the known structure.

Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis.

Polytheonamides are the most extensively modified ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) currently known. In RiPP biosynthesis, the processed peptide is usually released from a larger precursor by proteolytic cleavage to generate the bioactive terminal product of the pathway. For polytheonamides, which are members of a new RiPP family termed proteusins, we have recently shown that such cleavage is catalyzed by the cysteine protease PoyH acting on the precursor PoyA, both encoded in the polytheonamide biosynthetic gene cluster. We now report activity for PoyH under a variety of reaction conditions for different maturation states of PoyA and demonstrate a potential use of PoyH as a promiscuous protease to liberate and characterize RiPPs from other pathways. As a proof of concept, the identified recognition motif was introduced into precursors of the thiopeptide thiocillin and the lanthipeptide lichenicidin VK1, allowing for their site-specific cleavage with PoyH. Additionally, we show that PoyH cleavage is inhibited by PoyG, a previously uncharacterized chagasin-like protease inhibitor encoded in the polytheonamide gene cluster.

Polytheonamide biosynthesis showcasing the metabolic potential of sponge-associated uncultivated 'Entotheonella' bacteria.

The vast majority of microorganisms on the planet have not been grown under laboratory conditions due to unknown metabolic and environmental constraints. This uncultivated majority has enormous potential as a reservoir of unique enzymology and biosynthetic pathways. The following review offers a glimpse into this unexplored enzymatic stockpile through recent progress made on the biosynthesis of the potent polytheonamide cytotoxins. These structurally highly complex pore-forming peptides, isolated from the marine sponge Theonella swinhoei, are synthesized by the ribosome and then modified through numerous unusual transformations including iterative epimerase and N-methyltransferase activities. The bacterial source of these metabolites was identified as the taxonomically remote, uncultivated sponge symbiont 'Entotheonella factor' with a biosynthetic prowess that rivals those of industrially exploited microorganisms.

Targeting cholesterol in a liquid-disordered environment by theonellamides modulates cell membrane order and cell shape.

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3ß-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.

Interaction between the marine sponge cyclic peptide theonellamide A and sterols in lipid bilayers as viewed by surface plasmon resonance and solid-state (2)H nuclear magnetic resonance.

Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3ß-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid-state deuterium nuclear magnetic resonance ((2)H NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol % cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3α-cholesterol)-containing liposomes and pure POPC liposomes was comparably weak, confirmed the preference of the peptide for the 3ß-hydroxysterol-containing membranes. To further establish the formation of the complex of TNM-A with 3ß-hydroxysterols in lipid bilayers, solid-state (2)H NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The (2)H NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3ß-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3ß-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes.

Aurantoside J: a new tetramic acid glycoside from Theonella swinhoei. Insights into the antifungal potential of aurantosides.

The chemical investigation of an Indonesian specimen of Theonella swinhoei afforded four aurantosides, one of which, aurantoside J (5), is a new compound. The structure of this metabolite, exhibiting the unprecedented N-α-glycosidic linkage between the pentose and the tetramate units, has been determined through detailed spectroscopic analysis. The four obtained aurantosides have been tested against five fungal strains (four Candida and one Fusarium) responsible of invasive infections in immuno-compromised patients. The non-cytotoxic aurantoside I (4) was the single compound to show an excellent potency against all the tested strains, thus providing valuable insights about the antifungal potential of this class of compounds and the structure-activity relationships.

Koshikamide B, a cytotoxic peptide lactone from a marine sponge Theonella sp.

Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual amino acid residues. The unusual amino acids are N(delta)-carbamoylasparagine and 2-(3-amino-2-hydroxy-5-oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia cells and the human colon tumor (HCT-116) cell line with an IC50 value of 0.45 and 7.5 microg/mL, respectively.