Structure elucidation of phase I metabolites of the microtubule perturbagens: ceratamines A and B.

The heterocyclic alkaloids, ceratamines A and B, are isolates from a marine Pseudoceratina sp. sponge. They behave as antimitotic agents, with IC50 values in the low micromolar range. The mechanism of this activity involves the disruption of microtubule dynamics; therefore, the ceratamines are of great interest in cancer drug discovery. Studies of in vitro metabolism were performed using rat liver microsomes to begin to understand the pharmacokinetics of these unique natural products. A total of eight metabolites were identified using UV and LC-MS/MS techniques. The majority of metabolites were formed as a result of various demethylation reactions. The formation of two metabolites, M1 and M3, involved monooxygenation, most likely on the aromatic ring, however the exact structure has not been determined. UV absorbance revealed a hypsochromic shift as a result of monooxygenation, an observation that may suggest the loss of aromaticity; however, further investigation is required. The structures of two major metabolites of ceratamine B, M4 and M6, were confirmed by (1)H NMR spectroscopy. These metabolites formed as a result of demethylation at the methoxy and aminoimidazole, respectively.

Synthesis and DNA-binding affinity studies of glycosylated intercalators designed as functional mimics of the anthracycline antibiotics.

Anthracycline antibiotics such as daunomycin (Dauno) and doxorubicin (Dox) are well-known clinically used cancer chemotherapeutics, which, among other mechanisms, bind to DNA, thereby triggering a cascade of biological responses leading to cell death. However, anthracyclines are cardiotoxic, and drug resistance develops rapidly, thus limiting their clinical use. We report here the synthesis and DNA-binding affinity of a novel class of functional anthracycline mimetics consisting of an aromatic moiety linked to a carbohydrate (1-12). In the targets, the aromatic core consists of a 2-phenylbenzo[b]furan-3-yl, 2-phenylbenzo[b]thiophen-3-yl, 1-tosyl-2-phenylindol-3-yl, or 2-phenylindol-3-yl group that is bound to one of three aminosugars (daunosamine, acosamine, or 4-amino-2,3,4,6-tetradeoxy-alpha-l-hexopyranoside) via a propargyl linker. The DNA binding affinity of these twelve compounds has been evaluated by using both direct and indirect fluorescence measurements. Compared to Dauno and Dox, the DNA binding affinity of these analogues is weaker. However, both aromatic and aminosugar motifs are critical to DNA binding, with more influence coming from the structural features of the aromatic portion.

Dependence of DNA sequence selectivity and cell cytotoxicity on azinomycin A and B epoxyamide stereochemistry.

Evaluation of the importance of C18/C19 stereochemistry of azinomycin A/B epoxyamide partial structures with respect to DNA alkylation sequence selectivity is reported using a unique assay with a DNA oligomer containing imbedded normal (5'-GGC-3'/3'-CCG-5') and inverted (5'-CGG-3'/3'-GCC-5') azinomycin consensus cross-linking sequences. Both species were found to have unique selectivity profiles and alkylate DNA in a manner distinct from azinomycin B. Computational docking experiments support altered binding modes for the enantiomers.

Cellular effects induced by the antitumor agent azinomycin B.

Studies on the mechanism of action of the antitumor agent azinomycin B in vitro suggest that the drug elicits its lethal effects by the formation of interstrand crosslinks within the major groove of DNA. Here, we demonstrate the biological effects of the drug in vivo. Fluorescence imaging revealed localization of azinomycin B in the nuclear region of yeast. Moreover, experiments with oligonucleotide microarrays examined the effects of the drug across the yeast transcriptome. The results demonstrated a robust DNA damage response that supports the proposed role of the drug as a covalent DNA modifying agent. RT-PCR analysis validated the gene changes, and flow cytometry of azinomycin-treated yeast cells demonstrated a phenotypic S phase shift consistent with transcriptional effects.

Role of monovalent counterions in the ultrafast dynamics of DNA.

This paper examines the contribution of counterion motion to the electric-field dynamics in the interior of DNA. The electric field is measured by a coumarin fluorophore that is synthetically incorporated into an oligonucleotide, where it replaces a native base pair. The DNA is a 17-base-pair oligomer with no A- or G-tracts. Time-resolved Stokes-shift measurements on the coumarin are made from 40 ps to 40 ns with each of the alkali ions and or one of several tetraalkylammonium ions as the DNA counterion. With the possible exception of rubidium, there are no indications of site-specific binding of the counterions. For sodium and other ions with a smaller hydrodynamic radius, the dynamics are identical and are fit to a power law. For larger ions, there is a progressive increase in the rate of shifting after 1 ns. This effect correlates with the hydrodynamic radius of the counterion. The lack of change in the spectral shape of the emission shows that neither the broadly distributed power-law relaxation nor the extra nanosecond dynamics are due to heterogeneity in the relaxation rates of different helices.

Total synthesis of strobilurin B using a hetero-bis-metallated pentadiene linchpin.

An efficient, six-step stereocontrolled total synthesis of the antifungal agent strobilurin B is reported and was based on a convergent bi-directional coupling featuring a hetero-bis-1,4-metallated pentadiene system as the linchpin.

Effect of lesions on the dynamics of DNA on the picosecond and nanosecond timescales using a polarity sensitive probe.

This paper explores the effects of structural modifications on the fast dynamics of DNA and the ability of time-resolved Stokes shift spectroscopy to measure those changes. The time-resolved Stokes shift of a synthetic coumarin base-pair replacement within an oligomer is measured between 40 ps and 40 ns. Comparisons are made between 17mers without modification, with a deleted base near the coumarin and with the coumarin placed near the end of the oligomer. The deletion of a next-to-nearest-neighbor base pair does not change the subnanosecond dynamics, but does cause an additional motion with a time constant of approximately 20 ns. A candidate for this motion is the flipping of the abasic sugar out of the helix and the concomitant intrusion of water into the interior of the helix. A nearby chain end causes little change in the dynamics after 1 ns but leads to a reduction in the amplitude of the dynamics between 40 ps and 1 ns. We suggest that at the chain end, where DNA on one side of the probe has been replaced by water, the charge- stabilizing dynamics have the same overall amplitude, but that much of the relaxation occurs before the start of the measurement time window.

Tandem Stille/Suzuki-Miyaura coupling of a hetero-bis-metalated diene. Rapid, one-pot assembly of polyene systems.

[structure: see text]. The synthesis of a hetero-bis-metallo 1,3-butadiene is reported, and its use as an orthogonal Stille and Suzuki-Miyaura coupling partner is detailed. The tin/boron diene participated successfully in a one-pot, sequential Stille and Suzuki-Miyaura coupling protocol, and its utility was demonstrated in the two-step construction of the pentaene side chain of the Fusarium metabolite lucilactaene.

Asymmetric synthesis of the dibenzocyclooctadiene lignans interiotherin a and gomisin R.

[structure: see text] Asymmetric total syntheses of the dibenzocyclooctadiene lignans interiotherin A and angeloylgomisin R are reported. The syntheses were based on an atropdiastereoselective, copper-promoted biaryl coupling reaction, a diastereoselective hydroboration/Suzuki-Miyaura coupling reaction sequence, and an asymmetric boron-mediated tiglylation of an aryl aldehyde precursor.

DNA cross-linking by azinomycin B: Monte Carlo simulations in the evaluation of sequence selectivity.

A new set of charges specifically developed for biologically relevant N7-alkylated purine adducts have been implemented in the AMBER force field of the MacroModel package and applied to the conformational search of azinomycin B-DNA interactions. To perform a sequence dependent reactivity relationship study, four DNA triplets known to interact differently with the drug, 5'-GCT-3', 5'-GCC-3', 5'-GTC-3', and 5'-GTT-3', have been modeled in B-form and intercalative conformations. Monte Carlo simulations of all possible monoadducts and intercalative complexes have been carried out and analyzed using a filtering criterion that estimates the probability of covalent bond formation and covalent cross-linking. We observed a good correlation between existing experimental data and our computational estimations that validate the approach. The comparison of the conformational properties of the drug-DNA monoadducts and complexes confirms the most probable mechanism of action involving an initial aziridine and subsequent epoxide alkylation. The different hydrogen bond network in the monoadducts and in the intercalative complexes between the drug and the three base-pair receptor is the primary reason for the different cross-linking reactivity. In addition, steric hindrance of the major groove exposed methyl group of central thymine-based triplets plays an important role in the lack of the reactivity of these sequences. Synthetic work on the azinomycins and the information coming from this computational study will be important for the design of more potent or DNA sequence-selective agents based on the azinomycin skeleton.

Total synthesis of eupomatilones 4 and 6: structurally rearranged and atropisomerically fluxional lignan natural products.

[structure: see text] A convergent and diastereocontrolled total synthesis of eupomatilones 4 and 6 is reported and was based on a diastereoselective hydroboration/oxidation sequence and a convergent Lipshutz biarylcuprate cross-coupling reaction. The structure of eupomatilone 6 is revised.

Divergent and stereocontrolled synthesis of the enamide side chains of oximidines I/II/III, salicylihalamides A/B, lobatamides A/D, and CJ-12,950.

[reaction: see text] A unified strategy for the divergent and stereocontrolled introduction of the (E)- and (Z)-enamide side-chains of oximidines I, II, and III, salicylihalamides A and B, lobatamides A and D, and CJ-12,950 is detailed. The synthesis relied on the copper-promoted C-N coupling of (E)- and (Z)-vinyl iodides with a protected maleimide hemiaminal followed by deprotection and reaction of the resulting (E)- or (Z)-enelactam hemiaminals with O-methylhydroxylamine or propylidenetriphenylphosphorane.

Role of the azinomycin naphthoate and central amide in sequence-dependent DNA alkylation and cytotoxicity of epoxide-bearing substructures.

Studies report a strong correlation between duplex DNA alkylation and in vitro cytotoxicity for a series of azinomycin partial structures 2-6 bearing the biologically relevant epoxide. Compounds lacking the naphthoate ester (e.g., 5 and 6) were poorly reactive toward DNA and were biologically inactive, as were compounds bearing the naphthoate but lacking the terminal carboxamide (e.g., 2). Compounds were evaluated for cytotoxicity against two breast cancer cell lines. [structure: see text]

Structure-activity relationship studies of sulfonylpiperazine analogues as novel negative allosteric modulators of human neuronal nicotinic receptors.

Neuronal nicotinic receptors have been implicated in several diseases and disorders such as autism, Alzheimer's disease, Parkinson's disease, epilepsy, and various forms of addiction. To understand the role of nicotinic receptors in these conditions, it would be beneficial to have selective molecules that target specific nicotinic receptors in vitro and in vivo. Our laboratory has previously identified novel negative allosteric modulators of human α4ß2 (Hα4ß2) and human α3ß4 (Hα3ß4) nicotinic receptors. The effects of novel sulfonylpiperazine analogues that act as negative allosteric modulators on both Hα4ß2 nAChRs and Hα3ß4 nAChRs were investigated. This work, through structure-activity relationship (SAR) studies, describes the chemical features of these molecules that are important for both potency and selectivity on Hα4ß2 nAChRs.

A direct and efficient total synthesis of the tubulin-binding agents ceratamine A and B; use of IBX for a remarkable heterocycle dehydrogenation.

The total synthesis of the tubulin-binding agents ceratamine A and B is reported, along with des-methyl analogs, via a synthetic route that is high-yielding and operationally efficient. The synthetic route involved a Beckmann rearrangement to form an azepine ring precursor, a Knoevenagel condensation to install the benzylic side chain, and an effective imidazole annulation onto an alpha-aminoketone precursor with a protected S-methylisothiourea. Final dehydrogenation proved remarkably facile using IBX.

Nanoscale structure and dynamics of DNA.

DNA is depicted in elementary chemistry and biology texts as a perfect double helix; but local structural variations and nanoscale motions within the double helix are critical for its ability to be packaged, recognized, and transcribed. DNA is becoming a favored nanoscale assembly tool due to the precise pairing of complementary strands that in principle can bring nanoscale objects within a well-defined distance of each other. However, future nanotechnology applications of DNA need to take into account its variable nanoscale structural and dynamic properties, especially in terms of its solvent shell and counterions. This article highlights efforts of the authors to (1) interrogate nanoscale structures of DNA using nanoparticles and (2) measure the dynamic nature of DNA over six orders of magnitude in time, using a fluorescent reporter in the base stack.

Total synthesis of the eupomatilones.

Full details of the total syntheses of five members of the eupomatilone family of lignans are reported.

Synthesis of functional "top-half" partial structures of azinomycin a and B.

The design and synthesis of a detailed series of functional "top-half" substructures of azinomycin A and B is described.