Crystal structure of neocarzinostatin, an antitumor protein-chromophore complex.

Structures of the protein-chromophore complex and the apoprotein form of neocarzinostatin were determined at 1.8 angstrom resolution. Neocarzinostatin is composed of a labile chromophore with DNA-cleaving activity and a stabilizing protein. The chromophore displays marked nonlinearity of the triple bonds and is bound noncovalently in a pocket formed by the two protein domains. The chromophore pi-face interacts with the phenyl ring edges of Phe52 and Phe78. The amino sugar and carbonate groups of the chromophore are solvent exposed, whereas the epoxide, acetylene groups, and carbon C-12, the site of nucleophilic thiol addition during chromophore activation, are unexposed. The position of the amino group of the chromophore carbohydrate relative to C-12 supports the idea that the amino group plays a role in thiol activation.

Synthesis of (+)-dynemicin A and analogs of wide structural variability: establishment of the absolute configuration of natural dynemicin A.

BACKGROUND: Dynemicin A is an exceedingly potent antitumor antibiotic derived from microbial fermentation that cleaves double-stranded B-form DNA in vitro in the presence of activating factors such as NADPH or glutathione. Because of the structural complexity, high reactivity, and scarcity of natural dynemicin A, it has not been feasible to modify the structure to any significant extent. Previous studies have not determined the absolute configuration of the natural product. RESULTS: A multistep route for the preparation of enantiomerically pure, synthetic dynemicin A was developed. The absolute configuration of natural dynemicin was determined by comparing the synthetic drug with dynemicin A derived from fermentation. The route that was developed is highly convergent, as the result of a late-stage coupling reaction that combines two complex synthetic fragments, and has been shown to provide access to non-natural dynemicins of wide structural variability by modifications of these fragments. In this way, several nonnatural dynemicins, unavailable by any other means, were synthesized and shown to have DNA-cleaving activity in the presence of glutathione or NADPH. CONCLUSIONS: Enantiomerically pure dynemicin A is now available by laboratory synthesis. The natural, (+)-enantiomer of dynemicin A is shown to possess the 2S, 3S, 4S, 7R, 8R configuration. A wide variety of heretofore unavailable, active analogs of dynemicin A have been prepared and are found to produce subtle variations in sequence specificity of DNA cleavage compared to the natural product and, of potentially greater significance, display variations in the efficiency of DNA cleavage as a function of the activating agent.

In vivo distribution and activity of aphidicolin on dividing and quiescent cells.

In view of a possible use of aphidicolin, an inhibitor of DNA polymerases (including viral DNA polymerases), to control excessive cell proliferation we have investigated: the effect of the drug on the growth of several human neoplastic cells; the activity of synthetic analogs aimed at relating the structural feature of aphidicolin to cytotoxicity; the in vivo fate and distribution of aphidicolin in different fluids, organs and tissues of mice following parenteral and/or peroral administration.

One-step construction of the pentacyclic skeleton of saframycin A from a "Trimer" of alpha-amino aldehydes.

The entire skeleton of the saframycin antitumor antibiotics is assembled in one remarkable transformation (8 --> 9) from an N-linked oligomer of three alpha-amino aldehyde components, a reaction pathway that may parallel the biosynthetic route to the saframycins.

On the inherent instability of alpha-amino alpha'-fluoro ketones. Evidence for their transformation to reactive oxyvinyliminium ion intermediates.

[figure: see text] alpha-Amino alpha'-fluoro ketones are shown to be inherently unstable intermediates. Evidence is presented that they undergo enolization toward the amino group followed by expulsion of fluoride ion, forming a proposed oxyvinyliminium ion (amino-substituted oxyallyl cation). In protic, nucleophilic media the proposed intermediate is trapped by solvent. In the presence of a reactive diene, [4 + 3] cycloadducts have been isolated. Prior observations concerning fluorinated amino ketones are discussed in light of these findings.

Synthesis of C-protected alpha-amino aldehydes of high enantiomeric excess from highly epimerizable N-protected alpha-amino aldehydes.

A new procedure for the preparation of C-protected alpha-amino aldehydes of high enantiomeric excess is illustrated using five differently substituted alpha-(N-Fmoc)amino aldehydes as starting materials. Highly epimerization-prone substrates were converted to the corresponding morpholino nitrile-protected alpha-amino aldehydes with minimal racemization (products >/= 89% ee). Morpholino nitrile derivatives of phenylglycinal were crystallized and subjected to X-ray structural analysis, allowing for definitive determination of the stereochemistry of amino nitrile formation. A rationale for the stereoselectivity of amino nitrile formation is presented.

Synthesis of a broad array of highly functionalized, enantiomerically pure cyclohexanecarboxylic acid derivatives by microbial dihydroxylation of benzoic acid and subsequent oxidative and rearrangement reactions.

[reaction: see text]. We have found that the 1,2-dihydroxylation of benzoic acid with Alcaligenes eutrophus strain B9, first reported in 1971 by Reiner and Hegeman, is readily adapted for the preparation of tens to hundreds of grams of (1S,2R)-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylic acid of >95% ee. This unique substrate undergoes many specific oxidative and rearrangement processes. Among these are transformations of unanticipated chemical novelty and many products that have not been previously described.

Biomagnetic detection of gastric electrical activity in normal and vagotomized rabbits.

We recorded the vector magnetogastrogram (MGG) due to gastric electrical activity (GEA) in normal rabbits using a Superconducting QUantum Interference Device (SQUID) magnetometer and measured the degree of correlation of the MGG with 24 channels of serosal electrodes. The vector magnetometer allows us to non-invasively record three orthogonal magnetic field components and project the recorded magnetic field vector into arbitrary directions. We optimized the magnetic field vector direction to obtain the highest possible correlation with each serosal electrode recording. We performed a vagotomy and examined spatial and temporal changes in the serosal potential and in the transabdominal magnetic field. We obtained spatial information by mapping the recorded signals to the electrode positions in the gastric musculature. Temporal evidence of uncoupling was observed in spectral analyses of both serosal electrode and SQUID magnetometer recordings. We conclude that non-invasive recordings of the vector magnetogastrogram reflect underlying serosal potentials as well as pathophysiological changes following vagotomy.

DNA cleavage by an alpha, 3-dehydrotoluene precursor conjugated to a minor groove binding element.

The (Z)-allene-ene-yne 10, a precursor to a reactive alpha,3-dehydrotoluene biradical, was covalently linked to a minor groove binding element, and the resultant conjugate (9) was shown to bind and cleave DNA with sequence selectivity.

Gene transcription analysis of Saccharomyces cerevisiae exposed to neocarzinostatin protein-chromophore complex reveals evidence of DNA damage, a potential mechanism of resistance, and consequences of prolonged exposure.

The natural product neocarzinostatin (NCS), a protein-small molecule complex, exhibits potent antiproliferative activity in mammalian cells but has little apparent effect on the growth of the unicellular eukaryotic organism, Saccharomyces cerevisiae. Here, we show by whole-genome transcription profiling experiments that incubation of S. cerevisiae with NCS leads to dramatic and wide-ranging modifications in the expression profile of yeast genes. Approximately 18% of yeast transcripts are altered by 2-fold or more within 4 h of treatment with NCS. Analysis of the observed transcription profile provides evidence that yeast rapidly and continuously overexpress multiple DNA-damage repair genes during NCS exposure. Perhaps to meet the energetic requirements of continuous DNA-damage repair, yeast cells enter respiration upon prolonged exposure to NCS, although grown in nutrient-rich medium. The NCS protein component is readily transported into S. cerevisiae, as demonstrated by fluorescence microscopy of yeast treated with fluorescently labeled NCS. Transcription profiling experiments with neocarzinostatin protein alone implicate a specific resistance mechanism in yeast that targets the NCS protein component, one involving the nonclassical export pathway. These experiments provide a detailed picture of the effects of exposure to NCS upon yeast and the mechanisms they engage as a response to this protein-small molecule DNA-damaging agent.

Asymmetric synthesis of chiral organofluorine compounds: use of nonracemic fluoroiodoacetic acid as a practical electrophile and its application to the synthesis of monofluoro hydroxyethylene dipeptide isosteres within a novel series of HIV protease inhibitors.

Two stereoselective routes to a series of diastereomeric inhibitors of HIV protease, monofluorinated analogues of the Merck HIV protease inhibitor indinavir, are described. The two routes feature stereoselective construction of the fluorinated core subunits by asymmetric alkylation reactions. The first-generation syntheses were based on the conjugate addition of the lithium enolate derived from pseudoephedrine alpha-fluoroacetamide to nitroalkene 12, a modestly diastereoselective transformation. A more practical second-generation synthetic route was developed that is based on a novel method for the asymmetric synthesis of organofluorine compounds, by enolate alkylation using optically active fluoroiodoacetic acid as the electrophile in combination with a chiral amide enolate. Resolution of fluoroiodoacetic acid with ephedrine provides either enantiomeric form of the electrophile in > or = 96% ee. Alkylation reactions with this stable and storable chiral fluorinated precursor are shown to proceed in a highly stereospecific manner. With the development of substrate-controlled syn- or anti-selective reductions of alpha-fluoro ketones 44 and 45 (diastereomeric ratios 12:1-84:1), efficient and stereoselective routes to each of the four targeted inhibitors were achieved. The optimized synthetic route to the most potent inhibitor (syn,syn-4, K(i) = 2.0 nM) proceeded in seven steps (87% average yield per step) from aminoindanol hydrocinnamide 40 and (S)-fluoroiodoacetic acid, and allowed for the preparation of more than 1 g of this compound. The inhibition of HIV-1 protease by each of the fluorinated inhibitors was evaluated in vitro, and the variation of potency as a function of inhibitor stereochemistry is discussed.

A chiral N-crotonyloxazolidinone Diels-Alder adduct.

(4S)-4-Benzyl-3-[(4S,5S)-(1-methoxy-5-methylcyclohexen-4- yl)carbonyl]-2-oxazolidinone, C19H23NO4, M(r) = 329.40, monoclinic, P2(1), a = 11.453 (3), b = 7.163 (4), c = 11.929 (2) A, beta = 111.86 (2) degree, V = 908.3 (5) A3, Z = 2, D chi = 1.20 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.79 cm-1, F(000) = 352, T = 297 K, R = 0.034 for 885 reflections with Fo2 greater than 0. The molecule is extended in the crystal; there is a small twist, -13.1 (2) degree, about the amide-like C--N bond joining the oxazolidinone ring to the carbonyl group. The configurations at the two optical centers in the cyclohexene ring confirm the anticipated stereospecificity of the Diels-Alder cycloaddition synthesis.

Enzymatic activation of DNA cleavage by dynemicin A and synthetic analogs.

Dynemicin A (1), a member of the enediyne family of natural products, binds to double-stranded DNA (K(B) approximately 10(4) M(-1)) and in the presence of millimolar concentrations of a reducing cofactor such as NADPH or GSH reacts to cleave DNA. In this work, we show that the two flavin-based enzymes ferredoxin-NADP+ reductase and xanthine oxidase catalyze the reductive activation of 1 by NADPH and NADH, respectively. The enzyme-catalyzed reductive activation of 1 leads to more rapid and efficient cleavage of DNA, even with 10-20-fold lower concentrations of the stoichiometric reductant. Significantly, the enzymatic systems are also found to activate the tight-binding (K(B) > or = 10(6) M(-1)) synthetic dynemicin analogs 3 and 5 toward DNA cleavage. These same analogs do not undergo reductive activation with NADPH or NADH alone, where evidence has been obtained to support the proposal that the DNA-bound drugs are protected from reductive activation. The new enzymatic activation processes described may have important implications for chemistry occurring with 1 and synthetic analogs in vivo, as well as for the future development of dynemicin-based anticancer agents.