Cytochalasins from an Australian Marine Sediment-Derived Phomopsis sp. (CMB-M0042F): Acid-Mediated Intramolecular Cycloadditions Enhance Chemical Diversity

Chemical analysis of an Australian coastal marine sediment-derived fungus, Phomopsis sp. (CMB-M0042F), yielded the known cytochalasins J (<b>1</b>) and H (<b>2</b>), together with five new analogues, cytochalasins J₁–J₃ (<b>3</b>–<b>5</b>) and H₁ and H₂ (<b>6</b> and <b>7</b>). Structures of <b>1</b>–<b>7</b> were assigned on the basis of detailed spectroscopic analysis, chemical interconversion, and biosynthetic and mechanistic considerations. Of note, <b>1</b> and <b>2</b> proved to be highly sensitive to acid-mediated transformation, with <b>1</b> affording <b>3</b>–<b>5</b> and <b>2</b> affording <b>6</b> and <b>7</b>. Whereas <b>1</b>, <b>2</b>, <b>4</b>, and <b>5</b> were detected as natural products in crude culture extracts, <b>3</b>, <b>6</b>, and <b>7</b> were designated as acid-mediated handling artifacts. We propose novel stereo- and regiospecific intramolecular cycloadditions, under tight functional group control, that facilitate selective conversion of <b>1</b> and <b>2</b> to the rare 5/6/6/7/5- and 5/6/5/8-fused heterocycles <b>5</b> and <b>7</b>, respectively. Knowledge of acid sensitivity within the cytochalasin family provides a valuable cautionary lesson that has the potential to inform our analysis of past and future investigations into this structure class and inspire novel biomimetic transformations leading to new chemical diversity

Talarolide A, a Cyclic Heptapeptide Hydroxamate from an Australian Marine Tunicate-Associated Fungus, <i>Talaromyces</i> sp. (CMB-TU011)

A miniaturized 24-well plate microbioreactor approach was used to explore secondary metabolite media dependence in an Australian marine tunicate-associated fungus, <i>Talaromyces</i> sp. (CMB TU011). Detailed chemical investigations of an antifungal M1-saline cultivation yielded talarolide A (<b>1</b>), only the second reported natural cyclic peptide hydroxamate, and the first from a fungus. The antifungal properties of the M1-saline extract were attributed to the known diterpene glycoside sordarin (<b>2</b>). Structure elucidation of <b>1</b> and <b>2</b> was achieved by detailed spectroscopic analysis, with amino acid configurations in <b>1</b> assigned by the C₃ and C₁₈ Marfey’s methods, and l-Ala and d-Ala regiochemistry by the recently reported 2D C₃ Marfey’s method

FQE increases CpsD-P in <i>S. pneumoniae</i> D39.

<p><i>S. pneumoniae</i> D39 were grown to mid log phase in THY (OD₆₀₀ ≈ 0.35) and FQE at indicated concentrations were added. (A) These concentrations (µM) had no statistically significant effect on CFU/ml after 30, 60 and 120 mins. (B) Whole cell lysates were prepared from these cells, which were separated by SDS-PAGE and analyzed by immunoblotting using anti-CpsD, or anti-phosphotyrosine (to detect CpsD-P). (C) Densitometric analysis of CpsD-P from three separate experiments. The effect with addition of 5 µM was significantly higher than compared with 1.25 µM FQE (* - <i>P</i><0.05 by Student’s <i>t</i>-test). (D) For comparison, the effect of an in-frame <i>cpsB</i> deletion mutant on CpsD-P is shown.</p

Wollamides: Antimycobacterial Cyclic Hexapeptides from an Australian Soil <i>Streptomyces</i>

A soil <i>Streptomyces</i> nov. sp. (MST-115088) isolated from semiarid terrain near Wollogorang Station, Queensland, returned two known and two new examples of a rare class of cyclic hexapeptide, desotamides A and B (<b>1</b> and <b>2</b>) and E and F (<b>3</b> and <b>4</b>), respectively, together with two new d-Orn homologues, wollamides A and B (<b>5</b> and <b>6</b>). Structures were assigned by detailed spectroscopic and C₃ Marfey’s analysis. The desotamides/wollamides exhibit growth inhibitory activity against Gram-positive bacteria (IC₅₀ 0.6–7 μM) and are noncytotoxic to mammalian cells (IC₅₀ >30 μM). The wollamides exhibit antimycobacterial activity (IC₅₀ 2.8 and 3.1 μM), including reduction in the intracellular mycobacterial survival in murine bone marrow-derived macrophages

Screening of Marine Extract Library for inhibitors of CpsB activity.

<p>The ability of extracts to inhibit His₆CpsB dephosphorylation of <i>p</i>NPP in 1 M Tris pH 8.0 with 1 mM MnCl₂ was investigated in 96 well trays at 37°C. Shown is a plot of the two screening replicates reported as % phosphatase activity relative to the average of particular screening plate. The star represents the extract which produced the pure compound of interest.</p

FQE inhibits CpsB dephosphorylation of <i>p</i>NPP.

<p>(A) Structure of FQE which (B) inhibited CpsB dephosphorlyation of <i>p</i>NPP with IC₅₀ = 5.21 µM. (C) CpsB inhibition of pNPP dephosphorylation by FQE (10 µM) was investigated with increasing concentrations of CpsB^H5H7. Data shown is from three independent experiments (*** - <i>P</i><0.001 by Student’s <i>t</i>-test compared to no addition of CpsB^H5H7).</p

FQE also inhibits <i>E. coli</i> Wzb and CPS synthesis in <i>K. pneumoniae O1</i>.

<p>(A) His₆Wzb dephosphorylation of <i>p</i>NPP in presence of FQE in 1 M Tris pH 7.0 at 37°C. (B) Total CPS preparations from <i>K. pneumoniae</i> incubated with FQE were analysed by uronic acid assay. Data is from four independent experiments (20 µM vs 5 µM; * - <i>P</i><0.05 by Student’s <i>t</i>-test:).</p

FQE decreases capsule synthesis in <i>S. pneumoniae</i> D39.

<p>Total CPS preparations were isolated from equal numbers of bacteria after incubation with FQE for 1 h. CPS levels were analysed by either (A) uronic acid assay or alternatively (B) by separating CPS on SDS-PAGE, transferring to Nylon and the probing with α-cps2 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036312#s4" target="_blank">materials and methods</a>. Data in (A) is from ≥3 independent experiments (5 µM vs 1.25 µM; * - <i>P</i><0.05 by Student’s <i>t</i>-test).</p

Waspergillamide A, a Nitro <i>depsi</i>-Tetrapeptide Diketopiperazine from an Australian Mud Dauber Wasp-Associated <i>Aspergillus</i> sp. (CMB-W031)

Chemical profiling of extracts from a mud dauber wasp-associated fungus, <i>Aspergillus</i> sp. (CMB-W031), revealed a remarkably diverse array of secondary metabolites, with many biosynthetic gene clusters being transcriptionally responsive to specific culture conditions. Chemical fractionation of a jasmine rice cultivation yielded many known fungal metabolites, including the highly cytotoxic (−)-stephacidin B and an unprecedented nonribosomal peptide synthase derived nitro <i>depsi</i>-tetrapeptide diketopiperazine, waspergillamide A (<b>1</b>). All structures were assigned by detailed spectroscopic analysis and, where appropriate, chemical degradation and Marfey’s analysis

Trichodermides A–E: New Peptaibols Isolated from the Australian Termite Nest-Derived Fungus <i>Trichoderma virens</i> CMB-TN16

Chemical analysis of a fermentation of the Australian termite nest-derived fungus <i>Trichoderma virens</i> CMB-TN16 yielded five new acyclic nonapeptides, trichodermides A–E (<b>1</b>–<b>5</b>). Amino acid residues, configurations, and sequences were determined by a combination of spectroscopic (NMR and MS-MS) and chemical (C₃ Marfey’s) methods. The trichodermides adhere to the sequence homology pattern common to <i>Trichoderma</i> 11 amino acid residue peptaibols; however, unlike other peptaibols the trichodermides do not exhibit antibacterial or antifungal activity and exhibit low to no cytotoxicity against mammalian cells. This variability in biological activity highlights the importance of knowing both planar structures and absolute configurations when interpreting structure–activity relationships