Efficient solid-phase-based total synthesis of the bisintercalator TANDEM

[reaction: see text] In this article, the first solid-phase-based total synthesis of TANDEM, a synthetic analogue of triostin A, is described. In initial studies, the synthesis incorporated depsipeptide formation, introduction of chromophores, and disulfide bond formation on the solid phase, prior to a final solution-phase macrolactamization, to give the target molecule. Although pure TANDEM was obtained in an overall yield comparable to those for all syntheses to date, the yield of the final cyclization was low (11%). A more efficient approach involved removal from the solid phase prior to disulfide bond formation. The resulting linear peptide underwent macrolactamization under mild conditions and high dilution. Final disulfide bond formation was essentially quantitative and gave the target molecule, TANDEM, in an overall yield of 18%. The final compound was assessed for its ability to bind to 5'-TpA sequences on DNA by DNase I footprinting. This efficient synthesis sets the stage for a study of the structure-activity relationship of TANDEM and the natural product triostin A, with analogues containing "point mutations" at every site within the cyclic compounds.     

Genes involved in <Emphasis Type="Italic">Drosophila</Emphasis>glutamate receptor expression and localization

Background  

A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development.     

Tautomerism of 4-phenyl-2,4-dioxobutanoic acid. Insights from pH ramping NMR study and quantum chemical calculations

Aryldiketo acids (ADKs) exhibit the variety of biological activities, mainly due to large affinity toward divalent metal ions. Metal complexation ability of ADKs, as well as interactions with proteins, depend on tautomeric form present in solution. The main aim of this study was to fully explore the tautomeric preferences of 4-phenyl-2,4-dioxobutanoic acid (4PDA), as ADKs representative, in aqueous media at different pH values. 1D and 2D NMR spectroscopy in combination with quantum chemical calculations was applied in order to better understand the tautomeric preferences of 4PDA. The data in highly acidic media are especially interesting since there are no such findings in the literature due to low solubility of ADKs in molecular form. At low pH values, where 4PDA is unionized, the most abundant tautomeric form is enol with keto group closer to phenyl ring. At higher pH values, mixture of two 4PDA ionic forms coexists in solution. Their ratio calculated according to NMR data fits the values predicted using two experimentally determined pK _a values. Based on the complexity of ¹H NMR spectrum of monoanionic 4PDA form, coexistence of two stable rotamers was assumed. In an alkaline media, 4PDA is mostly present in dianionic form. As π-electrons of dianion are delocalized over an entire keto-enol moiety, spectral distinction between tautomers was not possible. Quantum chemical calculations were used to predict relative stability of tautomers. The predictions were in good accordance with experimental results only in case when explicit water molecule was included in calculations.     

Bonding preferences of C2X4-bridged bimetallic transition metal complexes of Ti, Cu, and Ag

The bonding preference of transition metal species of general formula [(PH3)2M]2(mu-C2X4), where M = Cu or Ag and X = O, S, Se, or Te, and (Cp2Ti)2(mu-C2X4), where X = S or Se, are explored using density functional theory. The relative energies of metal binding to the bridging ligand in a dithiolene-like vs dithiocarbamate-like manner are evaluated. In all cases, the most stable structure corresponds to dithiolene-like (or side-side) bonding, consistent with the vast majority of these compounds which have been experimentally characterized. However, for M = Ag and X = S, Se, or Te, the two isomers are nearly degenerate.     

Practical computational toolkits for dendrimers and dendrons structure design

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.     

Putative DNA quadruplex formation within the human c-kit oncogene

The DNA sequence, d(AGGGAGGGCGCTGGGAGGAGGG), occurs within the promoter region of the c-kit oncogene. We show here, using a combination of NMR, circular dichroism, and melting temperature measurements, that this sequence forms a four-stranded quadruplex structure under physiological conditions. Variations in the sequences that intervene between the guanine tracts have been examined, and surprisingly, none of these modified sequences forms a quadruplex arrangement under these conditions. This suggests that the occurrence of quadruplex-forming sequences within the human and other genomes is less than was hitherto expected. The c-kit quadruplex may be a new target for therapeutic intervention in cancers where there is elevated expression of the c-kit gene.     

Molecular Dynamics Simulations of Proteins with Chemically Modified Disulfide Bonds

Proteins that are used as therapeutic drugs act in the extracellular microenvironment. They usually have a small number of intramolecular disulfide bonds to help maintain their tertiary structure in the vascular circulation. In general, most cysteine residues are part of a disulfide bond with free sulfhydrals being uncommon. We have studied whether the site-specific chemical reduction of disulfides and the incorporation of a 3-carbon methylene bridge between the cysteines in interferon-α 2a would change the structure of this protein. Bridging of both of the disulfide bonds of interferon-α 2a was studied using two different molecular simulation protocols: (1) molecular dynamics, and (2) stochastic dynamics. We have shown that the disulfide bonds in interferon-α 2a can be reduced and chemically modified without significantly altering the tertiary structure of the protein. This offers the novel possibility of chemically modifying therapeutically important proteins without affecting their biological properties.     

Solid-phase synthesis of the cyclic peptide portion of chlorofusin, an inhibitor of p53-MDM2 interactions

The first solid-phase synthesis of the chlorofusin peptide is described. The synthesis involved side-chain immobilization of N(alpha)-Fmoc-Asp-ODmab. Synthesis of the linear peptide, initially incorporating racemic Ade8 and unsubstituted ornithine in place of the chromophore-bearing residue, was followed by cyclization on resin and peptide release to give a mixture of diastereomers. Resynthesis identified (by HPLC) the second isomer as analogous to the natural product. Initial biological assays, using an immunofluorescence method, suggest that the compounds are not cytotoxic but do not inhibit the p53/mdm2 interaction. [structure: see text]