Mitochondrial interaction of fibrosis-protective 5-methoxy tryptophan enhances collagen uptake by macrophages.

5-methoxy tryptophan (5-MTP) is an anti-fibrotic metabolite made by fibroblasts and epithelial cells, present in a micromolar concentrations in human blood, and is associated with the progression of fibrotic kidney disease, but the mechanism is unclear. Here, we show by microscopy and functional assays that 5-MTP influences mitochondria in human peripheral blood monocyte-derived macrophages. As a result, the mitochondrial membranes are more rigid, more branched, and are protected against oxidation. The macrophages also change their metabolism by reducing mitochondrial import of acyl-carnitines, intermediates of fatty acid metabolism, driving glucose import. Moreover, 5-MTP increases the endocytosis of collagen by macrophages, and experiments with inhibition of glucose uptake showed that this is a direct result of their altered metabolism. However, 5-MTP does not affect the macrophages following pathogenic stimulation, due to 5-MTP degradation by induced expression of indole-amine oxygenase-1 (IDO-1). Thus, 5-MTP is a fibrosis-protective metabolite that, in absence of pathogenic stimulation, promotes collagen uptake by anti-inflammatory macrophages by altering the physicochemical properties of their mitochondrial membranes.

Shedding light on the mitochondrial matrix through a functional membrane transporter.

The first fluorescent probes that are actively channeled into the mitochondrial matrix by a specific mitochondrial membrane transporter in living cells have been developed. The new functional probes (BCT) have a minimalist structural design based on the highly efficient and photostable BODIPY chromophore and carnitine as a biotargeting element. Both units are orthogonally bonded through the common boron atom, thus avoiding the use of complex polyatomic connectors. In contrast to known mitochondria-specific dyes, BCTs selectively label these organelles regardless of their transmembrane potential and in an enantioselective way. The obtained experimental evidence supports carnitine-acylcarnitine translocase (CACT) as the key transporter protein for BCTs, which behave therefore as acylcarnitine biomimetics. This simple structural design can be readily extended to other structurally diverse starting F-BODIPYs to obtain BCTs with varied emission wavelengths along the visible and NIR spectral regions and with multifunctional capabilities. BCTs are the first fluorescent derivatives of carnitine to be used in cell microscopy and stand as promising research tools to explore the role of the carnitine shuttle system in cancer and metabolic diseases. Extension of this approach to other small-molecule mitochondrial transporters is envisaged.

Stereochemical and Steric Control of Photophysical and Chiroptical Properties in Bichromophoric Systems.

Stereochemical and steric control of the relative spatial arrangement of the chromophoric units in multichromophoric systems offers an interesting strategy for raising unusual and appealing light-induced emission states. To explore and exploit this strategy, a series of conformationally restricted boron-dipyrromethene (BODIPY) dimers were designed by using tartaric acid as a symmetrical connector between the boron atoms of the dyes. The variety of stereoisomeric forms available for this bis(hydroxy acid) allows the relative spatial orientation of the chromophoric units in the dimer to be modified, which thus opens the door to modulation of the photophysical and chiroptical properties of the new bichromophoric systems. Chromophore alkylation introduces an additional level of control through distance-dependent steric interactions between the BODIPY units in the dimer, which also modulates their relative spatial disposition and properties.

Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes.

Tunneling nanotubes (TnTs) are thin channels that temporally connect nearby cells allowing the cell-to-cell trafficking of biomolecules and organelles. The presence or absence of TnTs in human neoplasms and the mechanisms of TnT assembly remains largely unexplored. In this study, we have identified TnTs in tumor cells derived from squamous cell carcinomas (SCC) cultured under bi-dimensional and tri-dimensional conditions and also in human SCC tissues. Our study demonstrates that TnTs are not specific of epithelial or mesenchymal phenotypes and allow the trafficking of endosomal/lysosomal vesicles, mitochondria, and autophagosomes between both types of cells. We have identified focal adhesion kinase (FAK) as a key molecule required for TnT assembly via a mechanism involving the MMP-2 metalloprotease. We have also found that the FAK inhibitor PF-562271, which is currently in clinical development for cancer treatment, impairs TnT formation. Finally, FAK-deficient cells transfer lysosomes/autophagosomes to FAK-proficient cells via TnTs which may represent a novel mechanism to adapt to the stress elicited by impaired FAK signaling. Collectively, our results strongly suggest a link between FAK, MMP-2, and TnT, and unveil new vulnerabilities that can be exploited to efficiently eradicate cancer cells.

Efficient multi-click approach to well-defined two-faced octasilsesquioxanes: the first perfect Janus nanocube.

The preparation of the first structurally well-defined Janus nanocube showing two chemically distinct opposed faces is described. The synthetic approach is based on a highly efficient and symmetry-controlled CuAAC functionalization of an octa-azido cubic silsesquioxane with a conformationally constrained tetra-alkyne with an appropriate spatial orientation of the triple bonds.

Nonafluorobutanesulfonyl azide as a shelf-stable highly reactive oxidant for the copper-catalyzed synthesis of 1,3-diynes from terminal alkynes.

Nonafluorobutanesulfonyl azide is a highly efficient reagent for the copper-catalyzed coupling of terminal alkynes to give symmetrical and unsymmetrical 1,3-diynes in good to excellent yields and with good functional group compatibility. The reaction is extremely fast (<10 min), even at low temperature (−78 °C), and requires substoichiometric amounts of a simple copper(I) or copper(II) salt (2­5 mol %) and an organic base (0.6 mol %). A possible mechanistic pathway is briefly discussed on the basis of model DFT theoretical calculations. The quantitative assessment of the safety of use and shelf stability of nonafluorobutanesulfonyl azide has confirmed that this reagent is a superior and safe alternative to other electrophilic azide reagents in use today.

Rhodium-catalyzed intermolecular C-H amination of simple hydrocarbons using the shelf-stable nonafluorobutanesulfonyl azide.

A new procedure has been developed for the direct intermolecular C-H amination of simple hydrocarbons using shelf-stable nonafluorobutanesulfonyl azide in the presence of a dirhodium(II) tetracarboxylate catalyst under mild reaction conditions. Some mechanistic details are briefly discussed on the basis of control experiments.

Controlled click-assembly of well-defined hetero-bifunctional cubic silsesquioxanes and their application in targeted bioimaging.

A general procedure for the assembly of hetero-bifunctional cubic silsesquioxanes with diverse functionality and a perfectly controlled distribution of functional groups on the inorganic framework has been developed. The method is based on a two-step sequence of mono- and hepta-functionalization through the ligand-accelerated copper(I)-catalyzed azide-alkyne cycloaddition of a readily available octaazido cubic silsesquioxane. The stoichiometry of the reactants and the law of binomial distribution essentially determine the selectivity of the key monofunctionalization reaction when a copper catalyst with strong donor ligands is used. The methodology has been applied to the preparation of a set of bifunctional nano-building-blocks with orthogonal reactivity for the controlled assembly of precisely defined hybrid nanomaterials and a fluorescent multivalent probe for application in targeted cell-imaging. The inorganic cage provides an improved photostability to the covalently attached dye as well as a convenient framework for the 3D multivalent display of the pendant epitopes. Thus, fluorescent bioprobes based on well-defined cubic silsesquioxanes offer interesting advantages over more conventional fully organic analogues and ill-defined hybrid nanoparticles and promise to become powerful tools for the study of cell biology and for biomedical applications.

Click assembly of dye-functionalized octasilsesquioxanes for highly efficient and photostable photonic systems.

New hybrid organic-inorganic dyes based on an azide-functionalized cubic octasilsesquioxane (POSS) as the inorganic part and a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BDP) chromophore as the organic component have been synthesized by copper(I)-catalyzed 1,3-dipolar cycloaddition of azides to alkynes. We have studied the effects of the linkage group of BDP to the POSS unit and the degree of functionalization of this inorganic core on the ensuing optical properties by comparison with model dyes. The high fluorescence of the BDP dye is preserved in spite of the linked chain at its meso position, even after attaching one BDP moiety to the POSS core. The laser action of the new dyes has been analyzed under transversal pumping at 532 nm in both the liquid phase and when incorporated into solid polymeric matrices. The monosubstituted new hybrid dye exhibits high lasing efficiency of up to 56 % with high photostability, with its laser output remaining at the initial value after 4×10(5) pump pulses in the same position of the sample at a repetition rate of 30 Hz. However, functionalization of the POSS core with eight fluorophores leads to dye aggregation, as quantum mechanical simulation has revealed, worsening the optical properties and extinguishing the laser action. The new hybrid systems based on dye-linked POSS nanoparticles open up the possibility of using these new photonic materials as alternative sources for optoelectronic devices, competing with dendronized or grafted polymers.

Octakis(3-azidopropyl)octasilsesquioxane: a versatile nanobuilding block for the efficient preparation of highly functionalized cube-octameric polyhedral oligosilsesquioxane frameworks through click assembly.

A one-step synthesis of octakis(3-azidopropyl)octasilsesquioxane from commercially available octakis(3-aminopropyl)octasilsesquioxane has been developed through a highly efficient diazo-transfer reaction under very mild conditions. Nonaflyl azide is shown to be a safer, cheaper, and more efficient reagent for this transformation than the better known and generally used diazo-transfer reagent triflyl azide. Octakis(3-azidopropyl)octasilsesquioxane is an excellent nanobuilding block that can be readily octafunctionalized with a range of terminal alkynes by copper(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition to provide new functional nanocages, maintaining a perfect 3D cubic symmetry. The mildness, simplicity, and efficiency of this approach have been demonstrated in the preparation of a glyco-polyhedral oligosilsesquioxane (POSS) conjugate and a BODIPY-POSS cluster (BODIPY = boron dipyrromethene).

Phthalimides as exceptionally efficient single electron transfer acceptors in reductive coupling reactions promoted by samarium diiodide.

Experimental and theoretical evidence shows that phthalimides are highly efficient single electron transfer acceptors in reactions promoted by samarium diiodide, affording ketyl radical anion intermediates, which participate in high-yielding inter- and intramolecular reductive coupling processes with different radicophiles including imides, oxime ethers, nitrones, and Michael acceptors.

Exploring the use of conformationally locked aminoglycosides as a new strategy to overcome bacterial resistance.

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.

A simple structural-based approach to prevent aminoglycoside inactivation by bacterial defense proteins. Conformational restriction provides effective protection against neomycin-B nucleotidylation by ANT4.

Herein, we describe how the conformational differences exhibited by aminoglycosides in the binding pockets of the ribosome and those enzymes involved in bacterial resistance can be exploited in the design of new antibiotic derivatives with improved activity in resistant strains. The simple modification shown in the figure, leading to the conformationally restricted 5, provides an effective protection against aminoglycoside inactivation by Staphylococcus aureus ANT4, both in vivo and in vitro.

Ketone-imide versus ketone-oxime reductive cross-coupling promoted by samarium diiodide: new mechanistic insight gained from a failed aminocyclopentitol synthesis.

[reaction: see text] The intramolecular 1,6-ketone/imide reductive coupling promoted by samarium diiodide competes favorably with an alternative 1,5-ketone/oxime ether coupling in a keto-oxime substrate derived from D-glucosamine N-protected with a phthalimido group. This pinacol coupling reaction affords new homochiral alpha-hydroxylactam scaffolds that could be useful in diversity-oriented synthesis. A mechanistic proposal for this reaction that explains the experimental results is supported by DFT quantum-mechanical calculations on model compounds.

Synthesis, inhibition properties, and theoretical study of the new nanomolar trehalase inhibitor 1-thiatrehazolin: towards a structural understanding of trehazolin inhibition.

A new trehazolin analogue, 1-thiatrehazolin, has been synthesized from carbohydrate precursors by a highly efficient route based on our previously developed ketone/oxime ether reductive carbocyclization reaction for the construction of the cyclitol ring and an intramolecular nucleophilic displacement reaction for the construction of the thiazoline ring. 1-Thiatrehazolin is a very potent, slow, tight-binding trehalase inhibitor. A structural model for trehalase inhibition by trehazolin and its analogues, based on the experimental results and supported by theoretical calculations, is proposed.

Hydrogen-bonding cooperativity: using an intramolecular hydrogen bond to design a carbohydrate derivative with a cooperative hydrogen-bond donor centre.

Neighbouring groups can be strategically located to polarise HO.OH intramolecular hydrogen bonds in an intended direction. A group with a unique hydrogen-bond donor or acceptor character, located at hydrogen-bonding distance to a particular OH group, has been used to initiate the hydrogen-bond network and to polarise a HO.OH hydrogen bond in a predicted direction. This enhanced the donor character of a particular OH group and made it a cooperative hydrogen-bond centre. We have proved that a five-membered-ring intramolecular hydrogen bond established between an amide NH group and a hydroxy group (1,2-e,a), which is additionally located in a 1,3-cis-diaxial relationship to a second hydroxy group, can be used to select a unique direction on the six-membered-ring intramolecular hydrogen bond between the two axial OH groups, so that one of them behaves as an efficient cooperative donor. Talose derivative 3 was designed and synthesised to prove this hydrogen-bonding network by NMR spectroscopy, and the mannopyranoside derivatives 1 and 2 were used as models to demonstrate the presence in solution of the 1,2-(e,a)/five-membered-ring intramolecular hydrogen bond. Once a well-defined hydrogen-bond is formed between the OH and the amido groups of a pyranose ring, these hydrogen-bonding groups no longer act as independent hydrogen-bonding centres, but as hydrogen-bonding arrays. This introduces a new perspective on the properties of carbohydrate OH groups and it is important for the de novo design of molecular recognition processes, at least in nonpolar media. Carbohydrates 1-3 have shown to be efficient phosphate binders in nonpolar solvents owing to the presence of cooperative hydroxy centres in the molecule.

Synthesis of the aminocyclopentitol moieties of the hopanoids of Zymomonas mobilis and 'Anacystis montana'.

The first synthesis of the cyclopentitol units in bacterial hopanoids has been accomplished from D-glucosamine and the possible biological activity of the free cyclitols as glycosidase inhibitors has been studied.

Experimental evidence for the existence of non-exo-anomeric conformations in branched oligosaccharides: the neomycin-B case.

For the first time in natural O-glycosides, a large amount of non-exo-anomeric conformation is experimentally detected, in solution.