Antonio Vargas-Berenguel

Ferrocene–β‐Cyclodextrin Conjugates: Synthesis, Supramolecular Behavior, and Use as Electrochemical Sensors

Ferrocene with a beta-cyclodextrin unit bound to one or both cyclopentadienyl rings through the secondary face were conveniently synthesized by regiospecific copper(I)-catalyzed cycloaddition of 2-O-propargyl-beta-cyclodextrin to azidomethyl or bis(azidomethyl)ferrocene. The supramolecular behavior of the synthesized conjugates in both the absence and presence of bile salts (sodium cholate, deoxycholate, and chenodeoxycholate) was studied by using electrochemical methods (cyclic and differential pulse voltammetry), isothermal titration calorimetry, and NMR spectroscopy (PGSE, CPMG, and 2D-ROESY). These techniques allowed the determination of stability constants, mode of inclusion, and diffusion coefficients for complexes formed with the neutral and, in some cases, the oxidized states of the ferrocenyl conjugates. It was found that the ferrocenyl conjugate with one beta-cyclodextrin unit forms a redox-controllable head-to-head homodimer in aqueous solution. The ferrocene-bis(beta-cyclodextrin) conjugate is present in two distinguishable forms in aqueous solution, each one having a different half-wave oxidation potential for the oxidation of the ferrocene. By contrast, only one distinguishable form for the oxidized state of the ferrocene-beta-cyclodextrin conjugate is detectable. The redox-sensing abilities of the synthesized conjugates towards the bile salts were evaluated based on the observed guest-induced changes in both the half-wave potential and the current peak intensity of the electroactive moiety.

Supramolecular Chemistry of Carbohydrate Clusters with Cores having Guest Binding Abilities

This review concentrates on both the protein receptor and guest binding abilities of carbohydrate clusters based on a cyclodextrin core. The combination of both complexation abilities is the basis of one of the pursued approaches for developing site-specific drug delivery systems. Influence on the molecular recognition properties of the number of appended saccharides, the type of carbohydrate clustering and the type of the spacer arms, among other factors, are discussed.

β-Cyclodextrin-Bearing Gold Glyconanoparticles for the Development of Site Specific Drug Delivery Systems

Three novel gold nanoparticles containing multiple long, flexible linkers decorated with lactose, β-cyclodextrin, and both simultaneously have been prepared. The interaction of such nanoparticles with β-d-galactose-recognizing lectins peanut agglutinin (PNA) and human galectin-3 (Gal-3) was demonstrated by UV-vis studies. Gal-3 is well-known to be overexpressed in several human tumors and can act as a biorecognizable target. This technique also allowed us to estimate their loading capability toward the anticancer drug methotrexate (MTX). Both results make these glyconanoparticles potential site-specific delivery systems for anticancer drugs.

Synthesis of Cluster N‐Glycosides Based on a β‐Cyclodextrin Core

A convenient method for the synthesis of cluster glycopyranosylamines based on β-cyclodextrin is presented (see diagram).

Dendritic Galactosides Based on a β‐Cyclodextrin Core for the Construction of Site‐Specific Molecular Delivery Systems: Synthesis and Molecular Recognition Studies

In order to evaluate the ability of multivalent glycosides based on a beta-cyclodextrin core as site-specific molecular carriers, a study on both the inclusion complexation behaviour and lectin binding affinity of branched and hyperbranched beta-cyclodextrins is presented. A series of cluster galactosides constructed on beta-cyclodextrin scaffolds containing seven 1-thio-beta-lactose or beta-lactosylamine bound to the macrocyclic core through different spacer arms were synthesised. In addition, the first synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing fourteen 1-thio-beta-D-galactose, 1-thio-beta-lactose and 1-thio-beta-melibiose residues was performed. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalenesulfonate (ANS) and 2-naphthalenesulfonate, and lectin from peanut (Arachis hypogaea) (PNA). The persubstitution of the primary face of the beta-cyclodextrin with saccharides led to a slight increase of the binding constant values for the inclusion complexation with ANS relative to the native beta-cyclodextrin. However, the increase of the steric congestion due to the presence of the saccharide residues on the narrow rim of the beta-cyclodextrin may cause a decrease of the binding ability as shown for sodium 2-naphthalenesulfonate. The spacer arms are not passive elements and influence the host binding ability according to their chemical nature. PNA forms soluble cross-linked complexes with cluster galactosides and lactosides scaffolded on beta-cyclodextrin but not with cluster galactopyranosylamines or melibiose. Both, perbranched and hyperbranched beta-cyclodextrins, form stronger complexes with PNA than the monomeric analogues. However, the use of hyperbranched CDs does not contribute to the improvement of the complex stability relative to heptakis-glycocyclodextrin derivatives. Finally, a titration experiment with PNA and a complex formed by a heptakis lactose beta-cyclodextrin derivative with sodium 2-naphthalenesulfonate showed the formation of a soluble cross-linked complex with stronger affinity constant and higher stoichiometry than those observed for the complex formation of PNA with the same heptakis-lactose beta-cyclodextrin derivative, suggesting the formation of a three component complex.

Convenient synthesis of O-(2-acitamido-2-deoxy-β-D-glucopyranosyl)-serine and -threonine building blocks for solid-phase glycopeptide assembly

The suitably protected building blocks for solid-phase glycopeptide synthesis, Nα-Fmoc-Ser(Ac3-β-D-GlcNAc)-OPfp, 11, and Nα-Fmoc-Ther(Ac3-β-D-GlcNAc)-OPfp, 12, have been synthesized by stereo-selective glycosidation of the 2-allyloxycarbonylamino glycosyl donor 7 with Nα-Fmoc-Ser -OPfp, 3, and Nα-Fmoc-Thr -OPfp, 4, followed by Pdo-catalysed allyl transfer from the N-allyloxycarbonyl group in the presence of acetic anhydride.

Influence of the H-site residue 108 on human glutathione transferase P1-1 ligand binding: Structure-thermodynamic relationships and thermal stability

The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (Kd ∼ 0.5 μM) when compared with those of the parent compounds, K  dEA ∼ 13 μM, K  dGSH ∼ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔCp values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.

β-Cyclodextrin dimers linked through their secondary faces with rigid spacer arms as hosts for bile salts.

A convenient synthesis of β-cyclodextrin dimers in which the two cyclodextrin units are linked by rigid tethers of relatively short length through their secondary sides is reported. Compounds hexa-2,4-diynediyl- and 1,4-phenylenediethyne-briged β-cyclodextrin dimers are obtained in good yields from mono-2-O-propargyl-β-cyclodextrin through Pd-mediated oxidative homo- and heterocoupling reactions. Isothermal titration calorimetry and NMR spectroscopy (PGSE and 2D-ROESY) are used to determine the thermodynamic parameters (K, ΔH, and TΔS°) for the complexation of such β-cyclodextrin dimers with sodium cholate, deoxycholate, and chenodeoxycholate as well as to estimate the size of the supramolecular structures. The binding of bile salts is enhanced relative to that of native β-cyclodextrin. Although chenodeoxycholate salt binds in a 1:1 fashion, cholate and deoxycholate salts bind in a 1:2 sequential mode.

Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S‐transferase (GST P1‐1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr‐108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys‐47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X‐ray diffraction. Surprisingly, in the absence of Cys‐47, Cys‐101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys‐47. The Cys‐47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G‐site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H‐site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H‐site and in the water network at the dimer interface. Copyright

Poly(amido amine)-based mannose-glycodendrimers as multielectron redox probes for improving lectin sensing.

An easy-to-prepare series of electroactive poly(amido amine) (PAMAM)-based dendrimers of generations G0 to G2 having mannopyranosylferrocenyl moieties in the periphery to detect carbohydrate-protein interactions is reported. The synthesis involved the functionalization of the PAMAM surface with azidomethylferrocenyl groups and subsequent coupling of mannoside units by the Cu(I)-catalyzed Huisgen reaction. The binding affinity of the series of electroactive glycodendrimers was studied by isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Upon complexation of the glycodendrimers conjugates with prototypical concanavalin A (Con A), voltammograms showed a decrease of the peak current. Such dendrimers showed a notable improvement of redox sensing abilities toward Con A when compared with mono- and divalent analogues, based on both the glycoside multivalent and ferrocene dendritic effects.