Aromatic Interactions in Glycochemistry: From Molecular Recognition to Catalysis.

Aromatic platforms are ubiquitous recognition motifs occurring in protein carbohydrate- binding domains (CBDs), RNA receptors and enzymes. They stabilize the glycoside/ receptor complexes by participating in stacking CH/π interactions with either the α- or ß- face of the corresponding pyranose units. In addition, the role played by aromatic units in the stabilization of glycoside cationic transition states has started being recognized in recent years. Extensive studies carried out during the last decade have allowed the dissection of the main contributing forces that stabilize the carbohydrate/aromatic complexes, while helping delineate not only the standing relationship between the glycoside/ aromatic chemical structures and the strength of this interaction but also their potential influence on glycoside reactivity.

Single-Step Glycosylations with 13 C-Labelled Sulfoxide Donors: A Low-Temperature NMR Cartography of the Distinguishing Mechanistic Intermediates.

Glycosyl sulfoxides have gained recognition in the total synthesis of complex oligosaccharides and as model substrates for dissecting the mechanisms involved. Reactions of these donors are usually performed under pre-activation conditions, but an experimentally more convenient single-step protocol has also been reported, whereby activation is performed in the presence of the acceptor alcohol; yet, the nature and prevalence of the reaction intermediates formed in this more complex scenario have comparatively received minimal attention. Herein, a systematic NMR-based study employing both 13 C-labelled and unlabelled glycosyl sulfoxide donors for the detection and monitoring of marginally populated intermediates is reported. The results conclusively show that glycosyl triflates play a key role in these glycosylations despite the presence of the acceptor alcohol. Importantly, the formation of covalent donor/acceptor sulfonium adducts was identified as the main competing reaction, and thus a non-productive consumption of the acceptor that could limit the reaction yield was revealed.

Peptide-biphenyl hybrid-capped AuNPs: stability and biocompatibility under cell culture conditions.

In this study, we explored the biocompatibility of Au nanoparticles (NPs) capped with peptide-biphenyl hybrid (PBH) ligands containing glycine (Gly), cysteine (Cys), tyrosine (Tyr), tryptophan (Trp) and methionine (Met) amino acids in the human hepatocellular carcinoma cell line Hep G2. Five AuNPs, Au[(Gly-Tyr-Met)2B], Au[(Gly-Trp-Met)2B], Au[(Met)2B], Au[(Gly-Tyr-TrCys)2B] and Au[(TrCys)2B], were synthesised. Physico-chemical and cytotoxic properties were thoroughly studied. Transmission electron micrographs showed isolated near-spherical nanoparticles with diameters of 1.5, 1.6, 2.3, 1.8 and 2.3 nm, respectively. Dynamic light scattering evidenced the high stability of suspensions in Milli-Q water and culture medium, particularly when supplemented with serum, showing in all cases a tendency to form agglomerates with diameters approximately 200 nm. In the cytotoxicity studies, interference caused by AuNPs with some typical cytotoxicity assays was demonstrated; thus, only data obtained from the resazurin based assay were used. After 48-h incubation, only concentrations ≥50 µg/ml exhibited cytotoxicity. Such doses were also responsible for an increase in reactive oxygen species (ROS). Some differences were observed among the studied NPs. Of particular importance is the AuNPs capped with the PBH ligand (Gly-Tyr-TrCys)2B showing remarkable stability in culture medium, even in the absence of serum. Moreover, these AuNPs have unique biological effects on Hep G2 cells while showing low toxicity. The production of ROS along with supporting optical microscopy images suggests cellular interaction/uptake of these particular AuNPs. Future research efforts should further test this hypothesis, as such interaction/uptake is highly relevant in drug delivery systems.

Therapeutic inhibition of pro-inflammatory signaling and toxicity to staphylococcal enterotoxin B by a synthetic dimeric BB-loop mimetic of MyD88.

Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88(-/-) mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1ß, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1ß production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.

A small molecule that mimics the BB-loop in the Toll interleukin-1 (IL-1) receptor domain of MyD88 attenuates staphylococcal enterotoxin B-induced pro-inflammatory cytokine production and toxicity in mice.

Toxic shock syndrome (TSS) is a clinical consequence of the profound amplification of host pro-inflammatory cytokine signaling that results from staphylococcal enterotoxin (SE) exposure. We recently reported that MyD88(-/-) mice were resistant to SEA or SEB toxic shock and displayed reduced levels of pro-inflammatory cytokines in their serum. Here we report that SEB stimulation of total mononuclear cells up-regulated MyD88 in monocytes and T cells. Further, MyD88 gene silencing in primary human cells using siRNA prevented SEB or SEB plus lipopolysaccharide (LPS) induction of interleukin-1ß (IL-1ß) transcriptional activation, suggesting that MyD88-mediated signaling is an essential component of SEB toxicity. We synthesized small molecules that mimic the conserved BB-loop in the Toll/IL-1 receptor (TIR) domain of MyD88. In primary human cells, these mimetics attenuated SEB-induced pro-inflammatory cytokine production. SEB stimulation of primary cells with mimetic affected newly synthesized MyD88 and downstream signaling components. Furthermore, LPS-induced MyD88 signaling was likewise inhibited in a cell-based reporter assay. More importantly, administration of mimetic reduced cytokine responses and increased survivability in a murine SEB challenge model. Collectively, these results suggest that MyD88 BB-loop mimetics interfere with SEB-induced pro-inflammatory signaling and toxicity, thus offering a potential approach in the therapy of toxic shock.

Preparation and characterization of gold nanoparticles capped by peptide-biphenyl hybrids.

Gold nanoparticles were prepared using peptide-biphenyl hybrids (PBHs) as capping agents. AuNPs were characterized by different techniques including UV-Vis, TEM, EDX, FT-IR, elemental analysis, (1)H NMR and (13)C CP/MAS NMR spectroscopy. TEM analysis showed that AuNPs present diameters in the range of 1.8-3.7 nm, depending on the structure and the amount of the capping PBH used. FT-IR spectroscopy and solid-state (13)C NMR revealed that the carboxylic group of PBHs, especially in the case of the acid ligands, interacts with the gold surface (in the form of carboxylate). The results confirm that PBHs are excellent stabilizers of AuNPs, being one of the first examples on the use of peptidomimetics-gold hybrid materials.

Biological and chemical studies on aryl hydrocarbon receptor induction by the p53 inhibitor pifithrin-α and its condensation product pifithrin-ß.

AIMS: Pifithrin α (PFTα), an inhibitor of the p53 protein, is regarded as a lead compound for cancer and neurodegenerative disease therapy. There is some evidence that this compound activates the aryl hydrocarbon receptor (AhR) in a complete independent way of the p53 inhibition and that it is easily converted to its condensation product pifithrin ß (PFTß). The aim of this study was to explore the ability of PFTα and of PFTß to induce a variety of AhR mediated processes. MAIN METHODS: Computational analysis using quantum chemical calculations and chemical analysis have been used to study the conformation of the compounds as well as the cyclization reaction. The AhR mediated processes of these compounds have been studied in a rainbow trout cell line (RTG-2) and in a rat hepatoma cell line (H4IIE). KEY FINDINGS: PFTα molecule could not take a planar conformation required for AhR activation whereas PFTß showed a conformation similar to those of the prototypical AhR ligand ß-naphthoflavone. In both cell lines, PFTα and PFTß provoked different responses related with AhR activation. However, when cyclization of PFTα to PFTß was hampered by acetylation of the exocyclic nitrogen, all these responses were not observed. These results lead to the conclusion that the activation of the AhR is probably caused by PFTß instead of PFTα. SIGNIFICANCE: Since PFTα is a promising compound for the development of new pharmaceuticals inhibiting p53, the chemical instability of this compound as well as the capacity of its transformation product should be taken into account.

Heterocyclic alpha-helix mimetics for targeting protein-protein interactions.

The design and synthesis of alpha-helix peptidomimetics using inverse electron demand Diels-Alder reactions is described. The potency of the resulting pyridazine-based library to disrupt the Bak/Bcl-X(L) interaction was tested using an in vitro fluorescence polarization assay.

A reversible reaction inside a self-assembled capsule.

Reversible encapsulation allows the direct observation of the isolated molecules under ambient conditions, at equilibrium and in the liquid phase. Here we show that capsules can amplify and stabilize molecules that are present in only trace concentrations in solution. Evidence is given that reversible chemical reactions take place within the capsule. Stabilization of reaction intermediates is a characteristic property of enzymes and is widely regarded as an essential feature of catalytic activity. Reactive molecules can also be stabilized by encapsulation, a process that involves completely surrounding the reactive species within synthetic receptors. Here, we show that self-assembled capsules can isolate and stabilize molecules that are present in only trace amounts in solution. The system amplifies the concentrations of high-energy species with reduced entropies.

MyD88-dependent and -independent signaling by IL-1 in neurons probed by bifunctional Toll/IL-1 receptor domain/BB-loop mimetics.

Interleukin (IL)-1beta is a pluripotent proinflammatory cytokine that signals through the type-I IL-1 receptor (IL-1RI), a member of the Toll-like receptor family. In hypothalamic neurons, binding of IL-1beta to IL-1RI mediates transcription-dependent changes that depend on the recruitment of the cytosolic adaptor protein myeloid differentiation primary-response protein 88 (MyD88) to the IL-1RI/IL-1 receptor accessory protein (IL-1RAcP) complex through homomeric Toll/IL-1 receptor (TIR)-TIR interactions. Through design and synthesis of bifunctional TIR mimetics that disrupt the interaction of MyD88 with the IL-1RI/IL-1RAcP complex, we analyzed the involvement of MyD88 in the signaling of IL-1beta in anterior hypothalamic neurons. We show here that IL-1beta-mediated activation of the protein tyrosine kinase Src depended on a MyD88 interaction with the IL-1RI/IL-1RAcP complex. The activation of the protein kinase Akt/PKB depended on the recruitment of the p85 subunit of PI3K to IL-1RI and independent of MyD88 association with the IL-1RI/IL-1RAcP complex. These bifunctional TIR-TIR mimetics represent a class of low-molecular-weight compounds with both an antiinflammatory and neuroprotective potential. These compounds have the potential to inhibit the MyD88-dependent proinflammatory actions of IL-1beta, while permitting the potential neuronal survival supporting actions mediated by the MyD88-independent activation of the protein kinase Akt.

Studies on aromatic compounds: inhibition of calpain I by biphenyl derivatives and peptide-biphenyl hybrids.

With the objective to understand structural features responsible for the biological activity, novel nonelectrophilic biphenyl derivatives and peptide-biphenyl hybrids have been synthesized and evaluated as calpain I inhibitors. The preliminary results indicate that the presence of additional aromatic rings (besides the biphenyl system) makes these compounds potent calpain inhibitors with IC50 values in the nanomolar range.

Peptide-biphenyl hybrids as calpain inhibitors.

Calpain is a cysteine protease that is activated by Ca2+. The over-activation of calpain, which occurs on increasing Ca2+ concentration, causes a variety of diseases. This paper reports experimental results on the inhibition of calpain I (mu-calpain) by peptide-biphenyl hybrids. We have found that some peptide-biphenyl hybrids, with aromatic amino acids in the peptide chains, inhibit calpain with IC50 values in the nanomolar range. Since the peptide-biphenyl hybrids reported in the present paper do not possess a reactive electrophilic functionality, we hypothesize that they interfere with the activation of calpain by Ca2+, and present experimental and computational results on the binding of peptide-biphenyl hybrids to Ca2+.

New oxazole-based peptidomimetics: useful building blocks for the synthesis of orthogonally protected macrocyclic scaffolds.

[structure: see text] The synthesis of a new family of densely functionalized oxazole-containing amino acids is described. These building blocks were employed for preparing macrocycles containing Lys and Glu residues by a combination of solid- and solution-phase synthesis. The resulting structures are presented as orthogonally protected scaffolds for supramolecular chemistry.