Wesley Y. Yoshida

New apratoxins of marine cyanobacterial origin from guam and palau

Two collections of the marine cyanobacterium Lyngbya sp. from Guam and Palau that both afforded the potent cytotoxin apratoxin A (1) each yielded different structural analogues with lower degrees of methylation. The new apratoxins, termed apratoxins B (2) and C (3), were evaluated for their in vitro cytotoxicity along with semisynthetic E-dehydroapratoxin A (4) to identify key structural elements responsible for the cytotoxicity and to initiate SAR studies on this novel family of depsipeptides. All analogues 2-4 displayed weaker cytotoxicity than 1, but to different extents. While compound 3 closely approached the cytotoxicity of 1, compounds 2 and 4 exhibited significantly reduced activity, possibly also related to a conformational change. The 16S rRNA genes of the different apratoxin producers have partially been sequenced and compared, and other genetic differences are currently being revealed.

Piperidine alkaloids from Piper methysticum

Pipermethystine (1), 3alpha,4alpha-epoxy-5beta-pipermethystine (2) and awaine (3) were isolated from the aerial parts of kava (Piper methysticum G. Forster, Piperaceae) and identified by HRMS and NMR spectroscopic analysis. 1 was concentrated in the stem peelings and leaves. 2 and 3 are new alkaloids with 2 found only in cv. Isa among the 11 cultivars examined, and 3 occurred primarily in young leaves of all cultivars. The stem peelings have been used in recent years as a source of kavalactones in kava dietary supplement industry. Quantitative aspects of these piperidine alkaloids in P. methysticum and their potential activities on human physiology are discussed.

Biosynthesis and Structure of Aeruginoside 126A and 126B, Cyanobacterial Peptide Glycosides Bearing a 2-Carboxy-6-Hydroxyoctahydroindole Moiety

Aeruginosins represent a group of peptide metabolites isolated from various cyanobacterial genera and from marine sponges that potently inhibit different types of serine proteases. Members of this family are characterized by the presence of a 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety. We have identified and fully sequenced a NRPS gene cluster in the genome of the cyanobacterium Planktothrix agardhii CYA126/8. Insertional mutagenesis of a NRPS component led to the discovery and structural elucidation of two glycopeptides that were designated aeruginoside 126A and aeruginoside 126B. One variant of the aglycone contains a 1-amino-2-(N-amidino-Delta(3)-pyrrolinyl)ethyl moiety at the C terminus, the other bears an agmatine residue. In silico analyses of the aeruginoside biosynthetic genes aerA-aerI as well as additional mutagenesis and feeding studies allowed the prediction of enzymatic steps leading to the formation of aeruginosides and the unusual Choi moiety.

Post‐translational Modification in Microviridin Biosynthesis

Cyanobacteria are prolific producers of bioactive natural products that mostly belong to the nonribosomal peptide and polyketide classes. We show here how a linear precursor peptide of microviridin K, a new member of the microviridin class of peptidase inhibitors, is processed to become the mature tricyclic peptidase inhibitor. The microviridin (mvd) biosynthetic gene cluster of P. agardhii comprises six genes encoding microviridin K, an apparently unexpressed second microviridin, two RimK homologues, an acetyltransferase, and an ABC transporter. We have over‐expressed three enzymes of this pathway and have demonstrated their biochemical function in vitro through chemical degradation and mass spectrometry. We show that a prepeptide undergoes post‐translational modification through cross‐linking by ester and amide bond formation by the RimK homologues MvdD and MvdC, respectively. In silico analysis of the mvd gene cluster suggests the potential for widespread occurrence of microviridin‐like compounds in a broad range of bacteria.

Structure and absolute configuration of karlotoxin-2, an ichthyotoxin from the marine dinoflagellate Karlodinium veneficum.

In an attempt to determine the cause of repeated fish kills in an estuarine aquaculture facility in Maryland, a toxin with hemolytic, cytotoxic, and ichthyotoxic properties, designated as karlotoxin-2 (KmTx2), was isolated from Karlodinium veneficum. The structure of KmTx2 was elucidated by means of detailed ID and 2D NMR spectra, including 2D INADEQUATE. The relative and absolute configurations of KmTx2 were determined using J-based configuration analysis and comparison of its degradation products with synthetic controls.

Polyalkylated cyclopentindoles: Cytotoxic fish antifeedants from a sponge, Axinella sp.

Abstract A Western Australian sponge, Axinella sp., yielded three polyalkylated indoles unsubstituted at C3, thus apparently not derived from tryptophan. The three compounds, herbindoles A, B, and C, are cytotoxic against KB cells and are fish antifeedants.

New polyoxygenated steroids exhibiting reversal of multidrug resistance from the gorgonian Isis hippuris

Abstract Eleven new ( 5–15 ) and one known ( 4 ) polyoxygenated steroids have been isolated from two collections of the gorgonian Isis hippuris and their structures determined by spectroscopic analysis and X-ray diffraction study. Except for 15 all others were polyoxygenated gorgosterols. X-Ray study of the previously reported compound 4 demonstrated the stereochemistry of the side chain moiety to be identical with that of gorgosterol. Compound 15 was a deacetyl derivative of the known steroid 16 having a spiroketal and a lactone function. The stereochemistry of 15 was confirmed by X-ray analysis, which also established the C-22 configuration of 16 which had remained unsolved. These compounds have been tested for reversal of multidrug resistance (MDR) with cancer cells expressing P-glycoprotein (P-gp). Some of them showed moderate activity.

Isolation and Structures of Nostopeptolides A1, A2 and A3 from the Cyanobacterium Nostoc sp. GSV224

Abstract The isolation and total structure determinations of nostopeptolides A1 (1), A2 (2) and A3 (3) are described. These cyclic depsipeptides, which are devoid of cytotoxic, antifungal and inhibition of protease activities, are characteristic constituents of the cryptophycin-producing cyanobacterium Nostoc sp. GSV224. Structure elucidation by NMR analysis was made more challenging by the existence of each nostopeptolide in three conformations. One-dimensional TOCSY experiments proved to be very useful in isolating and identifying the nine amino acid residues and the butyryl group in each compound. The absolute stereochemistries of 1–3 were determined by comparing the amino acids in the acid hydrolyzates directly with authentic standards.

The absolute stereochemistry of Kahalalide F

Abstract Kahalalide F ( 1 ) is a depsipeptide of 14 residues, five of which form a 19-membered ring. It was isolated from a marine mollusk, Elysia rufescens , and is currently in preclinical trials against lung and colon cancers. It was known from conventional amino acid analysis that five valine and two threonine residues represented D- and L- enantiomers, but their position in the molecule was not known. After extensive hydrolytic trials, a combination of acid hydrolysis and hydrazinolysis succeeded in definitive stereochemical assignment.

Halogenated Fatty Acid Amides and Cyclic Depsipeptides from an Eastern Caribbean Collection of the Cyanobacterium Lyngbya majuscula

A lipophilic extract of an eastern Caribbean collection of Lyngbya majuscula yielded two new halogenated fatty acid amides, grenadamides B (1) and C (2), and two new depsipeptides, itralamides A (3) and B (4), along with the known compounds hectochlorin and deacetylhectochlorin. The recently reported depsipeptide carriebowmide (5) was also present in the extract and isolated as its sulfone artifact (6). Compounds 1-4 were identified by spectroscopic methods. The configurations of the amino acid residues of 3, 4, and 6 were determined by LC-MS analyses of diastereomeric derivatives of the acid hydrolysates (advanced Marfeys method). Based on the configurational analysis of 6, in direct comparison with authentic carriebowmide (5), a minor structural revision of 5 is proposed. Compounds 1 and 2 displayed marginal activity against the beet armyworm (Spodoptera exigua). Compounds 1-4 and 6 were assessed for general cell toxicity in human embryonic kidney (HEK293) cells. Only itralamide B (4) displayed significant cytotoxicity, showing an IC(50) value of 6 +/- 1 muM.