Making epothilones fluoresce: design, synthesis, and biological characterization of a fluorescent N12-aza-epothilone (azathilone)

A green fluorescent 12-aza-epothilone (azathilone) derivative has been prepared through the attachment of the 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore to the 12-nitrogen atom of the azamacrolide core structure. While less potent than natural epothilones or different N12-acylated azathilone derivatives, NBD-azathilone (3) promotes tubulin assembly, inhibits cancer cell proliferation in vitro and arrests the cell cycle at the G2/M transition. Most significantly, the binding of 3 to cellular microtubules (MTs) could be directly visualized by confocal fluorescence microscopy. Based on competition binding experiments with laulimalide-stabilized MTs in vitro, the N12-Boc substituted azathilone 1, Epo A, and NBD-azathilone (3) all interact with the same tubulin-binding site. Computational studies provided a structural model of the complexes between beta-tubulin and 1 or 3, respectively, in which the NBD moiety of 3 or the BOC moiety of 1 directly and specifically contribute to MT binding. Collectively, these data demonstrate that the cellular effects of 3 and, by inference, also of other azathilones are the result of their interactions with the cellular MT network.

Synthesis and properties of isobicyclo-DNA

We present the synthesis of the isobicyclo-DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single isobicyclo-thymidine incorporations, fully modified A- and T-containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo-DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Isobicyclo-DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self-paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo-DNA is stable in fetal bovine serum and does not elicit RNaseH activity.

Lipid synthesis in protozoan parasites: a comparison between kinetoplastids and apicomplexans.

Lipid metabolism is of crucial importance for pathogens. Lipids serve as cellular building blocks, signalling molecules, energy stores, posttranslational modifiers, and pathogenesis factors. Parasites rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Here we discuss the tremendous recent advances that have been made in the understanding of the synthesis and uptake pathways for fatty acids and phospholipids in apicomplexan and kinetoplastid parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania. Lipid synthesis differs in significant ways between parasites from both phyla and the human host. Parasites have acquired novel pathways through endosymbiosis, as in the case of the apicoplast, have dramatically reshaped substrate and product profiles, and have evolved specialized lipids to interact with or manipulate the host. These differences potentially provide opportunities for drug development. We outline the lipid pathways for key species in detail as they progress through the developmental cycle and highlight those that are of particular importance to the biology of the pathogens and/or are the most promising targets for parasite-specific treatment.

Stereoselective synthesis and structure determination of a bicyclo[3.3.2]decapeptide

By analogy to the structural diversity of covalent bond networks between atoms within organic molecules, one can design topologically diverse peptides from mathematical graphs by assigning amino acids to graph nodes and peptide bonds to graph edges. The key is to use diamino acids or amino diacids as equivalents of trivalent graph nodes, which enables a variety of graph topologies beyond the standard linear and monocyclic graphs in natural peptides. Here the bicyclic decapeptide A1FGk2VFPE1AG2 (1b) was prepared and crystallized to assign its bridge stereochemistry. The bridge configuration appears as planned by the chirality of the branching amino acids. Bicyclization furthermore depends on the presence of matched chiralities in the branching amino acids. The stereoselective formation of the second bridge opens the way for the synthesis of a large family of bicyclic peptides as promising new scaffolds for drug design.

Convergent synthesis and cellular uptake of multivalent cell penetrating peptides derived from Tat, Antp, pVEC, TP10 and SAP

Cell penetrating peptides (CPP) are peptides of 10 to 30 residues derived from natural translocating proteins. Multivalency is known to enhance cellular uptake for the Tat peptide and closely related polycationic sequences. To test whether multivalency effects on cellular uptake might also occur with other CPP types, we prepared multivalent versions of the strongly cationic Tat, the amphipathic sequences Antp, pVEC and TP10, and the polyproline helix SAP by convergent thioether ligation of the linear CPP onto multivalent scaffolds, and evaluated their uptake in HeLa and CHO cells, intracellular localization, cytotoxicity and hemolysis. While multivalency did not increase the cellular uptake of pVEC or SAP, multivalency effects on uptake comparable to Tat were observed with TP10 and Antp, which are attributable to their polycationic nature. The efficient synthetic protocol for these divalent CPP and their localization in the cytoplasm suggest that CPP might be useful for application in cargo delivery into cells.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Central European hardwood trees in a high-CO 2 future: synthesis of an 8-year forest canopy CO 2 enrichment project

Rapidly increasing atmospheric CO2 is not only changing the climate system but may also affect the biosphere directly through stimulation of plant growth and ecosystem carbon and nutrient cycling. Although forest ecosystems play a critical role in the global carbon cycle, experimental information on forest responses to rising CO2 is scarce, due to the sheer size of trees. Here, we present a synthesis of the only study world-wide where a diverse set of mature broadleaved trees growing in a natural forest has been exposed to future atmospheric CO2 levels (c. 550ppm) by free-air CO2 enrichment (FACE). We show that litter production, leaf traits and radial growth across the studied hardwood species remained unaffected by elevated CO2 over 8years. CO2 enrichment reduced tree water consumption resulting in detectable soil moisture savings. Soil air CO2 and dissolved inorganic carbon both increased suggesting enhanced below-ground activity. Carbon release to the rhizosphere and/or higher soil moisture primed nitrification and nitrate leaching under elevated CO2; however, the export of dissolved organic carbon remained unaltered.Synthesis. Our findings provide no evidence for carbon-limitation in five central European hardwood trees at current ambient CO2 concentrations. The results of this long-term study challenge the idea of a universal CO2 fertilization effect on forests, as commonly assumed in climate-carbon cycle models.

Synthesis and triplex forming properties of pyrrolidino pseudoisocytidine containing oligodeoxynucleotides

Pyrrolidino pseudo-C-nucleosides are isosteres of natural deoxynucleosides which are protonated at the pyrrolidino ring nitrogen under physiological conditions. As constituents of a triplex forming oligodeoxynucleotide (TFO), the positive charge is expected to stabilise DNA triple helices via electrostatic interactions with the phosphodiester backbone of the target DNA. We describe the synthesis of the pyrrolidino isocytidine pseudonucleoside and the corresponding phosphoramidite building block and its incorporation into TFOs. Such TFOs show substantially increased DNA affinity compared to unmodified oligodeoxynucleotides. The increase in affinity is shown to be due to the positive charge at the pyrrolidino subunit

Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.