Triad of polar residues implicated in pH specificity of acidic mammalian chitinase.

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X-ray crystallography.

Evaluating indole-related derivatives as precursors in the directed biosynthesis of diazepinomicin analogues.

The effectiveness of precursor-directed biosynthesis to generate diazepinomicin (1) analogues with varied ring-A substitutents was investigated by feeding commercially available, potential ring-A precursors such as fluorinated tryptophans, halogenated anthranilates, and various substituted indoles into growing actinomycete culture DPJ15 (genus Micromonospora). Two new monofluorinated diazepinomicin analogues (2 and 3) were identified and characterized by spectroscopic methods. Both derivatives showed modest antibacterial activity against the Gram-positive coccus Staphylococcus aureus with MIC values in the range 8-32 microg/mL.

Probing natural product biosynthetic pathways using Fourier transform ion cyclotron resonance mass spectrometry.

Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous.

Biosynthesis of diazepinomicin/ECO-4601, a Micromonospora secondary metabolite with a novel ring system.

The novel microbial metabolite diazepinomicin/ECO-4601 (1) has a unique tricyclic dibenzodiazepinone core, which was unprecedented among microbial metabolites. Labeled feeding experiments indicated that the carbocyclic ring and the ring nitrogen of tryptophan could be incorporated via degradation to the 3-hydroxyanthranilic acid, forming ring A and the nonamide nitrogen of 1. Genomic analysis of the biosynthetic locus indicated that the farnesyl side chain was mevalonate derived, the 3-hydroxyanthranilic acid moiety could be formed directly from chorismate, and the third ring was constructed via 3-amino-5-hydroxybenzoic acid.

Reassessing the structure of pyranonigrin.

Fermentation extracts of the marine fungus Aspergillus niger LL-LV3020 were found to have relevant activity in a number of assays. Chemical screening of the extracts revealed that this organism produced numerous secondary metabolites in addition to its principal metabolite, citric acid. The compound with the most significant UV peak was isolated and its structure elucidated.

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes.

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.

Homolog separation, a necessity for the proper identification of fungal metabolites.

Monitoring of fungal extracts for the production of novel metabolites, using a modular analytical system combining HPLC with UV-MS-ELS detection, identified culture LL-W1278 as a fungus producing new biopolymers. Only a non-routine HPLC analysis of a culture extract revealed that the standard water-acetonitrile elution method did not separate all members of the metabolite complex. Fine-tuning the eluting solvents established that it was essential to include acid with the water-methanol system to separate the new materials.

Dioxapyrrolomycin biosynthesis in Streptomyces fumanus.

Streptomyces fumanus, intramurally coded as culture LL-F42248, produces a series of pyrrolomycins including dioxapyrrolomycin (1) as the principal component. Our biosynthetic studies revealed that feeding labeled acetate to growing cultures of S. fumanus yielded pyrrolomycins labeled in the phenyl ring only.

Fumaquinone, a new prenylated naphthoquinone from Streptomyces fumanus.

A new prenylated naphthoquinone antibiotic, fumaquinone (5,7-dihydroxy-2-methoxy-3-methyl-6-(3-methyl-but-2-enyl)[1,4]naphthoquinone) was isolated from cultures of Streptomyces fumanus (LL-F42248). Its chemical structure was determined primarily by NMR spectroscopy. Preliminary feeding experiments indicated the naphthoquinone is of polyketide origin, while the O-methyl and aromatic C-methyl groups are derived from methionine.

Absolute stereochemistry of unusual biopolymers from Ascomycete culture LL-W1278: examples that derivatives of (S)-6-hydroxymellein are also natural fungal metabolites.

The new linear polyesters, W1278-A, B, and C, were isolated from culture extracts of a freshwater Ascomycete fungus. Their structures were elucidated on the basis of spectroscopic analyses and chemical transformations, with the absolute configuration established by the circular dichroism (CD) method using (R)-(-)-6-hydroxymellein as reference. Unexpectedly, W1278-A, B, and C contain (S)-6-hydroxymellein residues as concluded from the display of the opposite ellipticity than that of known (3R)-(-)-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin as revealed by CD.

Additional pyrrolomycins from cultures of Streptomyces fumanus.

Along with dioxapyrrolomycin (1), four new pyrrolomycin antibiotics, namely, pyrrolomycin G (3), pyrrolomycin H (4), pyrrolomycin I (5), and pyrrolomycin J (6), were produced in cultures of Streptomyces fumanus. Apart from dioxapyrrolomycin, pyrrolomycin G and pyrrolomycin H are the only other chiral members of the pyrrolomycin family of antibiotics, and their absolute stereochemistry was deduced to be 13S. Here, we report the isolation, structure elucidation, and antimicrobial activity of these new pyrrolomycins.

Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp.

The structure of a new dibenzodiazepine alkaloid, diazepinomicin (1), isolated from the culture of a marine actinomycete of the genus Micromonospora was characterized using spectroscopic methods. Diazepinomicin represents a unique molecular class composed of a dibenzodiazepine core linked to a farnesyl side chain.

New biotransformation products of nemadectins.

Selected nemadectins (formerly LL-F28249 series) have been fed to a panel of microorganisms with the aim of generating new derivatives. In addition to products resulting from the oxidation of the terminal methyl group (C-29), a unique phosphorylated nemadectin was isolated. The phosphate group was determined to be at C-23 by HMBC between phosphorus and H-23.

Absolute stereochemistry of the strevertenes.

The CD exciton chirality method was applied to determine the absolute stereochemistry of the strevertenes, antifungal pentaene macrolides produced by Streptoverticillium sp. LL-30F848. The CD difference spectrum of strevertene A methyl ester 15-dimethylaminobenzoate showed a positive couplet between the dimethylaminobenzoate and the pentaene chromophores, and therefore established the 15R configuration.

Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256. II. Isolation and structure determination.

Fermentations of the marine fungus Hypoxylon oceanicum (LL-15G256) were found to have potent antifungal activity. Isolation and purification of the antifungal agents provided two classes of compounds, macrocyclic polylactones and the lipodepsipeptides 15G256 gamma (1), 15G256 delta (2) and 15G256 epsilon (3). The isolation and structure elucidation of the lipodepsipeptides, all containing D-glutamate, L-serine, and the rare amino acid beta-ketotryptohan, are described in this paper.

Isolation, characterization and structure of a new allenic polyine antibiotic produced by fungus LL-07F275.

Antibiotic 07F275 (1), produced by submerged fermentations of fungal culture LL-07F275, was isolated and characterized despite its inherent instability. Its UV spectrum was identical with that of nemotin, a member of the allenic polyacetylene family, but a molecular weight of 218 daltons indicated a new compound. Structure 1 was determined on the basis of spectroscopic evidence, particularly NMR.

Extracellular metal-free corrinoids from Rhodopseudomonas spheroides.

Rhodopseudomonas spheroides when grown on a medium deficient of cobalt excretes significant amounts of descobaltocorrinoids into the culture broth. If grown in the presence of 4 microM CoCl2 only intracellular cobalt-containing corrinoids are detected. The extracellular corrinoids have been identified as hydrogenobyrinic acid c-amide and hydrogenobyrinic acid a,c-diamide which are accompanied by varying amounts of the corresponding 13-epicorrinoids.