Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Isolation and identification of five impurities of ALB 109564(a) drug substance.

Four low-level impurities were detected during the development and scale up of the synthesis of ALB 109564(a). These impurities were isolated and characterized in order to determine how they originated in the drug substance. The information allowed the elimination of one impurity and a significant reduction in the relative abundance of the other three. A fifth impurity was detected in an accelerated stability study sample of the drug substance. The degradant was found to be the free acid resulting from the hydrolysis of the methyl ester within the indoline moiety of ALB 109564(a). The characterization of this impurity allowed for changes in the handling of the drug substance which minimized the formation of the impurity.

Gas-phase intramolecular elimination reaction studies of steviol glycosides in positive electrospray and tandem mass spectrometry.

This paper reports the first study of the gas-phase intramolecular elimination reaction of steviol glycosides in positive electrospray mass spectrometry. The observed glycosylated product ions are proposed to be formed via an intramolecular elimination of sugar units from the parent molecule ion. It was further proven by MS/MS studies and deuterium labeling experiments with one of the steviol glycosides, rebaudioside A. These mass spectrometric results confirmed that the new glycosylated product ions observed are most likely formed by the combination of glucose moieties (Glu) II-IV and Glu I via a gas-phase intramolecular elimination reaction.

Citreamicins with potent gram-positive activity.

Two new xanthone antibiotics, citreamicin delta (1) and epsilon (2), with potent activity against Gram-positive pathogens including multidrug-resistant Staphylococcus aureus (MDRSA) were discovered. Compounds 1 and 2 exhibited MIC values < 1 microg/mL versus a number of resistant strains. The compounds were obtained from EtOAc extracts of Streptomyces vinaceus and were purified by countercurrent chromatography and reversed-phase HPLC. Their structures were elucidated using primarily NMR and mass spectroscopy.

Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography-electrospray ionization mass spectrometry.

Unusual amino acids such as beta-methoxytyrosine (beta-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among beta-MeOTyr isomers was poor. The best separation of beta-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.

Gymnangiamide, a cytotoxic pentapeptide from the marine hydroid Gymnangium regae.

A cytotoxic aqueous extract from the marine hydroid Gymnangium regae provided a novel linear pentapeptide, designated gymnangiamide (1). The planar structure of 1 was elucidated by interpretation of spectral data as well as chemical degradation and derivatization studies. In addition to the amino acids isoleucine and phenylserine, this peptide contained N-desmethyldolaisoleuine, O-desmethyldolaproine, and alpha-guanidino serine, three residues that have not previously been reported in a natural product. The absolute configurations of the constituent amino/guanidino acids were determined by chemical degradation and derivatization, followed by HPLC and LC-MS comparison with authentic standards. Gymnangiamide (1) was moderately cytotoxic against a number of human tumor cell lines in vitro.

Cyclonellin, a new cyclic octapeptide from the marine sponge Axinella carteri.

Cyclonellin (1), a new cyclic octapeptide, was isolated from an aqueous extract of the marine sponge Axinella carteri. Its structure was elucidated by interpretation of NMR spectral data of the intact compound and N-terminal Edman sequencing of linear peptide fragments obtained by partial hydrolysis of 1. The absolute configurations of the constituent amino acids were determined by acid hydrolysis, derivitization with FDAA, and LC-MS analyses.

Caulibugulones A-F, novel cytotoxic isoquinoline quinones and iminoquinones from the marine bryozoan Caulibugula intermis.

An extract of the marine bryozoan Caulibugula intermis, collected in the Indo-Pacific off Palau, produced a distinct pattern of differential cytotoxicity in the National Cancer Institute's 60 cell line antitumor screen. Bioactivity-directed fractionation of the extract provided six new compounds, caulibugulones A-F (1-6). The structures of these novel metabolites were determined by spectrochemical analyses including LC-MS, HRFABMS, 1-D and 2-D NMR experiments, and by comparison with related compounds. The structures of compounds 2 and 3 were confirmed by chemical interconversion. The isolated compounds exhibited IC(50)'s of 0.03-1.67 microg/mL against murine tumor cells in an in vitro cytotoxicity assay.

Geographic distribution of three alkaloid chemotypes of Croton lechleri.

Three known alkaloids, isoboldine (2), norisoboldine (1), and magnoflorine (8), have been isolated for the first time from Croton lechleri, a source of the wound healing latex "sangre de grado". An HPLC system was developed, and a large number of latex and leaf samples of C. lechleri from 22 sites in northern Peru and Ecuador were analyzed to gain an understanding of the natural variation in alkaloid content for the species. Up to six alkaloids were found to occur in the leaves including, in addition to those listed above, thaliporphine (3), glaucine (4), and taspine (9), whereas the latex contained only 9. Taspine (9) is the component that has been previously found to be responsible for the wound healing activity of C. lechleri latex, and its mean concentration throughout the range examined was found to be 9% of the latex by dry weight. In addition, three chemotypes are defined based on the alkaloid content of the leaves, and the geographic distribution of these chemotypes is discussed along with a quantitative analysis of the alkaloid content as a function of chemotype.