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To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis.Materials & MethodsPreviously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid (RGD) were used as cell culture substrates to control cell adhesion at the nanoscale. We conducted in vitro chondrogenesis of mesenchymal stem cells on the nanopatterns. We evaluated cohesion and GJIC in cell condensates.ResultsMechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling.ConclusionCell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

This work describes the synthesis and properties of a dicyanomethylene‐substituted indolo[3,2‐b]carbazole diradical ICz‐CN. This quinoidal system dimerises almost completely to (ICz‐CN)2, which contains two long C(sp3)−C(sp3) σ‐bonds between the dicyanomethylene units. The minor open‐shell ICz‐CN component in the solid‐state mixture was identified by EPR spectroscopy. Cyclic voltammetry and UV–visible spectroelectrochemical data, as well as comparison with reference monomer ICz‐Br reveal that the nature of the one‐electron oxidation of (ICz‐CN)2 at ambient temperature and ICz‐CN at elevated temperature is very similar in all these compounds due to the prevailing localization of their HOMO on the ICz backbone. The peculiar cathodic behaviour reflects the co‐existence of (ICz‐CN)2 and ICz‐CN. The involvement of the dicyanomethylene groups stabilizes the close‐lying LUMO and LUMO+1 of (ICz‐CN)2 and especially ICz‐CN compared to ICz‐Br, resulting in a distinctive cathodic response at low overpotentials. Differently from neutral ICz‐CN, its radical anion and dianion are remarkably stable under ambient conditions. The UV/Vis(–NIR) electronic transitions in parent (ICz‐CN)2 and ICz‐CN and their different redox forms have been assigned convincingly with the aid of TD‐DFT calculations. The σ‐bond in neutral (ICz‐CN)2 is cleaved in solution and in the solid‐state upon soft external stimuli (temperature, pressure), showing a strong chromism from light yellow to blue–green. Notably, in the solid state, the monomeric diradical species is predominantly formed under high hydrostatic pressure (>1 GPa). The work at the University of Málaga was funded by the MICINN (PID2019-110305GB-I00, PID2019-104293GB-I00 and PCI2019-111825-2), Instituto de Salud Carlos III (ISCIII) (RETIC ARADYAL, RD16/0006/0012) and Junta de Andalucía (UMAFEDERJA-080, UMA-FEDERJA-007 and P09FQM-4708) projects. S.R.G. also thanks the Ayuda para la incorporación de doctores of the University of Malaga. The work at the University of Alicante was supported by the MICINN (PID2019-106114GB-I00) and Generalitat Valenciana (AICO/2018/175) projects. The work at SIOC was supported by National Natural Science Foundation of China (grants no. 21875279 and 21790362). The authors thankfully acknowledge the computer resources, technical expertise, and assistance provided by the SCBI (Supercomputing and Bioinformatics) center of the University of Malaga. The spectro-electrochemical studies (R.N.) were fully funded by Spectroelectrochemistry Reading (a spinout company of the University of Reading). M.P.-A. would like to acknowledge the support of the European Research Council (ERC) Grant “Hecate”, reference no. 695527, held by Prof. Graeme Ackland. This work has been also supported by MCIU/MINECO through the projects CTQ2013-48252-P, CTQ2015-67755-C02-01-R and PGC2018-094814-B-C21.

Effective cartilage development relies on the successful formation of mesenchymal cell condensates. Mesenchymal condensation is a prevalent morphogenetic transition, which involves the upregulation of the adhesive extracellular glycoprotein fibronectin (FN). During condensation, there is an active directional migration of cells from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers). In this study, we live imaged the first 40 h of mesenchymal condensation during chondrogenesis on nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), present in FN. Results show cell-substrate adhesions modulate both single-cell and collective cell migration during mesenchymal condensation. Single cell tracking analysis showed that substrate adhesion determines the migration mode, protrusion formation and the directionality of the cell movement. Cells on the more adhesive nanopatterns presented traits among amoeboid and mesenchymal modes of migration facilitating a more directional movement and reducing contact inhibition of locomotion (CIL), which allows merging and condensation. Inhibition experiments demonstrated that neural cadherin (N-Cad) is required in cell-cell interactions, enabling cells to coordinate their movement and directionality in a multicellular environment and to maintain the group cohesiveness during migration. Altogether, this contributes to create a sufficiently dynamic scenario, in which there is a balance between cell-substrate and cell-cell adhesions for condensates to grow. Our results provide a framework for the regulation of single and collective cell migration during mesenchymal condensation, through nanoscale cell-substrate adherence.Summary statementThe fine tuning of substrate adherence through nanopatterning allows control of mesenchymal cell migration and determines condensation during chondrogenesis in vitro.

Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests. Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry. The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4+ Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%. Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4+ Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T-cell proliferation compared to native CLV.

Herein, we present an easy and efficient synthesis of amino terminal dendrons, combining protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido-functionalized core, these dendrons lead to a more efficient and controlled convergent synthesis of dendrimers with different sizes and shapes and multivalence. The amino terminal dendrimers were analyzed by diffusion-ordered spectroscopy experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by molecular dynamic simulations. The construction of these kinds of nanostructures, in a simple and efficient way, opens new opportunities for biomedical applications. Moreover, by choosing the appropriate core, these versatile macromolecules become an excellent fluorescent biomarker.

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell&ndash, cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell&ndash, substrate adhesion in the tissue engineering strategies for cartilage repair.

Osseointegration of metal prosthetic implants is a yet unresolved clinical need that depends on the interplay between the implant surface and bone cells. The lack of a relationship between bone cells and metal has traditionally been solved by coating the former with &ldquo, organic&rdquo, ceramics, such as hydroxyapatite. A novel approach is hereby presented, immobilizing covalently dendrimeric structures onto titanium implants. Amide-based amino terminal dendrons were synthetized and coupled to titanium surfaces in a versatile and controlled way. The dendritic moieties provide an excellent scaffold for the covalent immobilization of bioactive molecules, such as extracellular matrix (ECM) protein components or antibiotics. Herein, tripeptide arginine-glycine-aspartic acid (RGD) motifs were used to decorate the dendritic scaffolds and their influence on cell adhesion and proliferation processes was evaluated.

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes We are thankful for the support given by the Spanish grants BFU2013-43513-R, SAF2016-76689-R, CTQ2015-70698-R, RTI2018-099877-B-I00, CTQ2013-49317-EXP, CTQ2013-41339-P, CTQ2016-75870-P, CTQ2015-71896-REDT (Red de Fotoquímica Biológica) and Instituto de Salud Carlos III (ISCIII; RETIC ARADYALRD16/0006/0012), Junta de Andalucía (PI0250-2016 and UMA18-FEDERJA-007), Orfeo-cinqa network CTQ2016-81797-REDC; the Xunta de Galicia (2015-CP082, ED431C 2017/19, Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund - ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. M.E.V. also acknowledges the support provided by the Fundación AECC (IDEAS197VAZQ grant) SI

Extracellular matrix remodeling plays a pivotal role during mesenchyme patterning into different lineages. Tension exerted from cell membrane receptors bound to extracellular matrix ligands is transmitted by the cytoskeleton to the cell nucleus inducing gene expression. Here, we used dendrimer-based arginine&ndash, glycine&ndash, aspartic acid (RGD) uneven nanopatterns, which allow the control of local surface adhesiveness at the nanoscale, to unveil the adhesive requirements of mesenchymal tenogenic and osteogenic commitments. Cell response was found to depend on the tension resulting from cell&ndash, substrate interactions, which affects nuclear morphology and is regulated by focal adhesion size and distribution.

Allergic drug reactions are currently a major public health problem affecting patient health and increasing healthcare costs. They are caused by interactions between a drug and the human immune system and result in symptoms ranging from urticaria or angioedema to those more serious such as anaphylaxis or anaphylactic shock. The most commonly accepted mechanism for immunological activation is based on the hapten hypothesis. Drugs are low molecular weight substances that cannot cause an immune response on their own. However, they can act as haptens and form covalent adducts with proteins. The resulting hapten-carrier (drug-protein) conjugate can induce the production of IgE antibodies or T cells. An epitope, or antigenic determinant, is the part of the drug-protein antigen that is specifically recognized by the immune system. This may involve not only the drug derivative but also part of the carrier protein. Understanding the way in which drugs are metabolized after protein conjugation is vital in order to make progresses in the diagnosis of clinical allergy. In this review, recent advances in the identification of the chemical structures of antigenic determinants involved in immediate allergic reactions to drugs are presented. We have focused on drugs that most commonly elicit these reactions: betalactam and quinolone antibiotics and the non-steroidal anti-inflamatory drug pyrazolone. This will be discussed from a chemical point of view, relating our understanding of drug structure, chemical reactivity and immune recognition.

Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells. During chondrogenic commitment, active extracellular matrix (ECM) remodeling takes place, playing an instructive role in the differentiation process. Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation, these cultures are rather limited with respect to their ability to interrogate cells in cell–ECM interactions. In the present study, the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration. The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance, constructed from the corresponding atomic force microscopy images, and correlated with the cell adhesion and differentiation response. The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment, and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation. Open image in new window

Aiming to address a stable chondrogenesis derived from mesenchymal stromal cells (MSCs) to be applied in cartilage repair strategies at the onset of osteoarthritis (OA), we analyzed the effect of arginine&ndash, glycine&ndash, aspartate (RGD) density on cell condensation that occurs during the initial phase of chondrogenesis. For this, we seeded MSC-derived from OA and healthy (H) donors in RGD-dendrimer-poly(L-lactic) acid (PLLA) nanopatterned substrates (RGD concentrations of 4 &times, 10&minus, 9, 10&minus, 8, 2.5 &times, 8, and 10&minus, 2 w/w), during three days and compared to a cell pellet conventional three-dimensional culture system. Molecular gene expression (collagens type-I and II&ndash, COL1A1 and COL2A1, tenascin-TNC, sex determining region Y-box9-SOX9, and gap junction protein alpha 1&ndash, GJA1) was determined as well as the cell aggregates and pellet size, collagen type-II and connexin 43 proteins synthesis. This study showed that RGD-tailored first generation dendrimer (RGD-Cys-D1) PLLA nanopatterned substrates supported the formation of pre-chondrogenic condensates from OA- and H-derived human bone marrow-MSCs with enhanced chondrogenesis regarding the cell pellet conventional system (presence of collagen type-II and connexin 43, both at the gene and protein level). A RGD-density dependent trend was observed for aggregates size, in concordance with previous studies. Moreover, the nanopatterns&rsquo, had a higher effect on OA-derived MSC morphology, leading to the formation of bigger and more compact aggregates with improved expression of early chondrogenic markers.

Dendrimeric Antigens (DeAns) consist of dendrimers decorated with multiple units of drug antigenic determinants. These conjugates have been shown to be a powerful tool for diagnosing penicillin allergy using in vitro immunoassays, in which they are recognized by specific IgE from allergic patients. Here we propose a new diagnostic approach using DeAns in cellular tests, in which recognition occurs through IgE bound to the basophil surface. Both IgE molecular recognition and subsequent cell activation may be influenced by the tridimensional architecture and size of the immunogens. Structural features of benzylpenicilloyl-DeAn and amoxicilloyl-DeAn (G2 and G4 PAMAM) were studied by diffusion Nuclear Magnetic Resonance (NMR) experiments and are discussed in relation to molecular dynamics simulation (MDS) observations. IgE recognition was clinically evaluated using the basophil activation test (BAT) for allergic patients and tolerant subjects. Diffusion NMR experiments, MDS and cellular studies provide evidence that the size of the DeAn, its antigen composition and tridimensional distribution play key roles in IgE-antigen recognition at the effector cell surface. These results indicate that the fourth generation DeAns induce a higher level of basophil activation in allergic patients. This approach can be considered as a potential complementary diagnostic method for evaluating penicillin allergy. Yes

Mesenchymal stem cells (MSCs) are promising candidates for a range of tissue regeneration applications. Adequate scaffolds are necessary for their application in vivo, where interactions between cells and the surface material are critical. Arginine-glycine-aspartic acid tripeptides (RGD) were conjugated to polyamidoamine (PAMAM) dendrimers and used to pre-treat test surfaces. We demonstrate that pre-treatment with dendrimer-presented tripeptides efficiently increases MSC adhesion to a polystyrene test surface, and that treatment effectiveness is related to how tripeptides are presented by the dendrimer to the cell. We tested both R-G-D–dendrimer and dendrimer–R-G-D arrangements and found the former to be optimal in terms of surface adhesion.

In vitro drug allergy tests have limited sensitivity, partly due to a poor understanding of the immunological recognition of in vitro drug-protein conjugates. We have designed and synthesized multivalent mono- and bi-epitope dendrimeric antigen (DeAn) conjugates and studied their chemical and tridimensional structures. We describe differences in the spatial distribution and conformation of these conjugated epitopes for the first time: a partially hidden benzylpenicilloyl and a more exposed amoxicilloyl. Our data suggest that DeAn conjugates provide a useful model for studying IgE recognition in patients who suffer from an allergic reaction to benzylpenicillin and/or amoxicillin. 1D and 2D NMR, MDS and immunochemical studies provide evidence that both antigen composition and tridimensional distribution play key roles in IgE-antigen recognition. Bi-epitope DeAn conjugates could potentially allow the diagnosis of patients allergic to any of these two drugs with a single test and represent the basis for a broadly-applicable in vitro assay. From the clinical editor: The prevalence of allergic drug reactions is rising and there is an imperative need to identify patients at risk. In this interesting and important article, the authors developed a novel method for detecting drug specific IgE antibodies, responsible for allergic reactions, by using multivalent mono- and bi-epitope Dendrimeric Antigen (DeAn) conjugates. The continued success of this research may pave way of eventual development of a simple diagnostic test.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

Over recent years the importance of dendrimers has continued to increase in both life and material sciences. Multiple chemical strategies have been described toward the preparation of dendrimeric structures. Here, we present a new approach for the production of all-aliphatic polyamide dendrimers. In our approach, iterative 3,3′-diaminopivalic acid connections act as building blocks for dendrimer construction. 3,3′-Diazidopivalic acid units underpin a two-step pathway involving carboxylic acid–amine condensation, followed by azide reduction. The carboxylic acid–amine condensation step can be carried out by conventional methods, while the hydrogen-catalyzed azide-to-amine reduction is a clean process particularly appropriate for dendrimer preparation. Spectroscopic characterization of functionalized surface azides (Gn-N3) and amines (Gn-NH2) confirm the proposed structures. These dendrimers were studied in explicit solvent by atomistic forcefield-based molecular dynamics to characterize structural properties such as shape, radius and monomer distribution. Our results show that these compounds are similar in size and shape, with Gn-NH2 having a slightly bigger size and exhibiting lower terminal unit backfolding.

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells. Open image in new window

Neutral water-soluble aza-dipyrromethene boron difluoride (aza-BODIPY) derivatives are synthesized for biological imaging applications. Aqueous solubility is increased by optimizing the synthetic conditions to obtain aza-BODIPYs with several tetraethyleneglycol groups. Their spectral properties and assays performed on cultured cells, demonstrate the potential of these compounds as near-infrared (NIR) emitting fluorescent probes.

Aminonaphthalimide-BODIPY energy transfer cassettes were found to show very fast (kEET ≈ 10(10)-10(11) s(-1) and efficient BODIPY fluorescence sensitization. This was observed upon one- and two-photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two-photon absorption cross-section δ of the dyads is significantly incremented by the presence of the aminonaphthalimide donor [δ ≈ 10 GM for the BODIPY versus 19-26 GM in the dyad at λ(exc)=840 nm; 1 GM (Goeppert-Mayer unit)=10(-50) cm(4) smolecule(-1) photon-(1)]. The electronic decoupling of the donor and acceptor, which is a precondition for the energy transfercassette concept, was demonstrated by time-dependent density functional theory calculations. The applicability of the new probes in the one- and twophoton excitation mode was demonstrated in a proof-of-principle approach in the fluorescence imaging of HeLa cells. To the best of our knowledge, this is the first demonstration of the merging of multiphoton excitation with the energy transfer cassette concept for a BODIPY-containing dyad.

34p.-5 fig.-1 tab., Background :Allergic reactions to β-lactams are amongst the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate., Methods: We have treated B-lymphocytes with either AX or a biotinylated analogue (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques., Results: We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B-lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins., Conclusions: These results constitute the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response., This work was supported by grants SAF2015-68590R, SAF2012-36519 from MINECO, Spain and RIRAAF/RD12/0013/0008 from ISCIII to D.P-S.; by grants CTQ2013-41339-P from MINECO, Spain and RD12/0013/0003 from ISCIII to E. P-I.; and by grants PI12/02529 and PI15/01206 from MINECO- grant cofunded by ERDF, Spain, RIRAAF/RD12/0013/0001 from ISCIII, CTS-06603 from Junta de Andalucía , grant cofunded by the ERDF, PI-0545-2010, PI-0551 and PI-0699-2011 from Junta de Andalucía to M.J.T and M.B.

A new fluorescent dendrimeric antigen (DeAn) based on a dendron with amoxicilloyl terminal groups was synthesized. The synthesis was carried out using a novel class of all-aliphatic polyamide dendrimer (BisAminoalkylPolyAmide Dendrimers, or BAPAD) involving the direct condensation of 3,3′-diazidopivalic acid as a building block. Iterative azide reduction/amide formation increases the dendrimer generation. The BAPAD dendrimer was designed with a cystamine core. Reduction of the disulfide bond allows the incorporation of BAPAD dendrons into a 1,8-naphthalimide functionalized with a maleimide group. The fluorescence properties of DeAn were studied in PBS and compared with the properties of an equivalent dendron possessing amino-terminal groups. Both molecules shown high fluorescence quantum yields in PBS and could readily be visualized by fluorescence microscopy. DeAn was used as a synthetic antigen in a biomedical assay that tests their potential as an amoxicillin carrier in drug internalization by dendritic cells (DC) from tolerant and allergic patients. Cytometry data suggest that the dendrons are non-toxic and easily internalized by DCs, while confocal microscopy images indicate that the compounds are preferentially accumulated in the cytoplasm. These results indicate that BAPAD dendrons are good candidates for synthetic scaffolds for biomedical applications.

A new series of all-heterahomocalixarene-type structures was prepared. The new hetera[2(n)]metacyclophanes obtained contain a benzophenone moiety as photoactive component. Although both forms possess identical building blocks and differ only in ring size, they are markedly different in photochemical reactivity. The cyclophane size is the driving force for the photochemical behaviour of the benzophenone.

The organizational effect induced by noncovalent interactions such as hydrogen-bonding (H-B) and metal ion complexation on photoinduced processes is discussed. These include the intermolecular photodimerization of cinnamates, which is shown to occur under topochemical control within the supramolecular assemblies, and the intramolecular photodimerization of a bis-anthracene receptor where photoregulation of the recognition event is achieved. Progress in using supramolecular interactions toward the preparation of assemblies promoting charge separation and charge transport in all-organic photovoltaic devices rests on the preparation of materials that are adapted to the requirements of solid-state devices.

Novel 2-(1-substituted-1H-1,2,3-triazol-4-yl)pyridine (pytl) ligands have been prepared by "click chemistry" and used in the preparation of heteroleptic complexes of Ru and Ir with bipyridine (bpy) and phenylpyridine (ppy) ligands, respectively, resulting in [Ru(bpy)(2)(pytl-R)]Cl-2 and [Ir(ppy)(2)-(pytl-R)]Cl (R=methyl, adamantane (ada), beta-cyclodextrin (beta CD)). The two diastereoisomers of the Ir complex with the appended beta-cyclodextrin, [Ir-(ppy)(2)(pytl-beta CD)]Cl, were separated. The [Ru(bpy)(2)(pytl-R)]Cl-2 (R=Me, ada or beta CD) complexes have lower lifetimes and quantum yields than other polypyridine complexes. In contrast, the cyclometalated Ir complexes display rather long lifetimes and very high emission quantum yields. The emission quantum yield and lifetime (Phi = 0.23, tau = 1000 ns) of [Ir(ppy)(2)(pytl-ada)]Cl are surprisingly enhanced in [Ir(ppy)(2)(pytl-beta CD)]Cl (Phi=0.54, tau = 2800 ns). This behavior is unprecedented for a metal complex and is most likely due to its increased rigidity and protection from water molecules as well as from dioxygen quenching, because of the hydrophobic cavity of the beta CD covalently attached to pytl. The emissive excited state is localized on these cyclometalating ligands, as underlined by the shift to the blue (450 nm) upon substitution with two electron-withdrawing fluorine substituents on the phenyl unit. The significant differences between the quantum yields of the two separate diastereoisomers of [Ir(ppy)(2)(pytl-beta CD)]Cl (0.49 vs. 0.70) are attributed to different interactions of the chiral cyclodextrin substituent with the A and A isomers of the metal complex.

Background Beta-lactam (BLs) antibiotics are among the drugs most frequently eliciting allergic reactions, thus posing an important clinical problem. Protein haptenation plays a key role in immunological reactions to BLs and this process is considered necessary for the activation of the immune system. Therefore, it is of interest to develop tools to monitor this process. We have synthesized a biotinylated amoxicillin derivative (AX-B) to be used in the characterization of protein haptenation by amoxicillin (AX). Methods AX-B was obtained binding a biotin moiety at the lateral chain of AX. The recognition of AX-B by IgE of allergic patients was analyzed by RAST inhibition. AX and AX-B were used for the in vitro modification of proteins in serum and in cell culture. Modified proteins were separated by SDS-PAGE and detected by Western blot using an anti-AX antibody (for AX-modification) or HRP-streptavidin (for AX-B-modification). Distribution of intracellular protein adducts with AX-B was analyzed by confocal microscopy. Results AX-B showed a chemical reactivity similar to AX and competition studies suggested that AX-B and AX can modify the same targets. AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with nonselective allergy to BLs, whereas competition by AX-B was poorer with sera of AX-selective patients. AX-B allowed the detection of protein adducts with high sensitivity. This derivative bound to all of the previously identified major AX targets in serum but made possible to detect some new targets. In addition, in cell culture, AX-B protein adducts could be detected intracellularly. Conclusions AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity. The identity of novel AX-B targets will be the subject of future studies.

Ruthenium metallosurfactant forms micelles that show efficient electronic energy-transfer processes in the presence of an analogous iridium complex. Modulation of the process is achieved upon incorporation of these amphiphiles into cetyltrimethylammonium bromide micelles, allowing a tuning of the two emissions.

The preparation of a novel fullerene-thiophene derivative by Diels-Alder addition of terthiophene S,S-dioxide was demonstrated. Extrusion of SO 2 from the adduct is an effective process that yields a stable cyclohexadiene-1,4-bisthiophene-C 60 adduct in good isolable yield. The product has been accurately characterized and opens the way to synthesize new C 60 derivatives "via" Diels-Alder methodology without the possibility of cycloreversion. Electrochemical and spectroscopic properties of this macromolecule were studied and supported by theoretical calculations to interpret its electronic structure. The first approach to the electropolymerization of this macromonomer produces donor-acceptor molecular wires providing a new and versatile way to fullerene-based double cable polymers.