Highly efficient grafting of hetero-complementary amidinium and carboxylate hydrogen-bonding/ionic pairs onto polymer surfaces.

We describe a grafting methodology, based on thiol-fluoroarene chemistry, to efficiently incorporate complementary hydrogen-bonding carboxylate and amidinium groups into polymer backbones. The process was optimized both in solution and on the surface of processed films, with the aim to produce materials showing hetero-complementary adhesion.

Multivalent Lactose-Ferrocene Conjugates Based on Poly (Amido Amine) Dendrimers and Gold Nanoparticles as Electrochemical Probes for Sensing Galectin-3.

Galectin-3 is considered a cancer biomarker and bioindicator of fibrosis and cardiac remodeling and, therefore, it is desirable to develop convenient methods for its detection. Herein, an approach based on the development of multivalent electrochemical probes with high galectin-3 sensing abilities is reported. The probes consist of multivalent presentations of lactose-ferrocene conjugates scaffolded on poly (amido amine) (PAMAM) dendrimers and gold nanoparticles. Such multivalent lactose-ferrocene conjugates are synthesized by coupling of azidomethyl ferrocene-lactose building blocks on alkyne-functionalized PAMAM, for the case of the glycodendrimers, and to disulfide-functionalized linkers that are then used for the surface modification of citrate-stabilized gold nanoparticles. The binding and sensing abilities toward galectin-3 of both ferrocene-containing lactose dendrimers and gold nanoparticles have been evaluated by means of isothermal titration calorimetry, UV-vis spectroscopy, and differential pulse voltammetry. The highest sensitivity by electrochemical methods to galectin-3 was shown by lactosylferrocenylated gold nanoparticles, which are able to detect the lectin in nanomolar concentrations.

Synthesis of Phosphoramidite Monomers Equipped with Complementary Bases for Solid-Phase DNA Oligomerization.

We describe the preparation of two monomers that bear complementary nucleobases at the edges (guanine-2'-deoxycytidine and 2-aminoadenine-2'-deoxyuridine) and that are conveniently protected and activated for solid-phase automated DNA synthesis. We report the optimized synthetic routes leading to the four nucleobase derivatives involved, their cross-coupling reactions into dinucleobase-containing monomers, and their oligomerization in the DNA synthesizer.

Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence.

Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHHn- for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-Hisn tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.

Peptide Half-Life Extension: Divalent, Small-Molecule Albumin Interactions Direct the Systemic Properties of Glucagon-Like Peptide 1 (GLP-1) Analogues.

Noncovalent binding of biopharmaceuticals to human serum albumin protects against enzymatic degradation and renal clearance. Herein, we investigated the effect of mono- or divalent small-molecule albumin binders for half-life extension of peptides. For proof-of-principle, the clinically relevant glucagon-like peptide 1 (GLP-1) was functionalized with diflunisal, indomethacin, or both. In vitro, all GLP-1 analogues had subnanomolar GLP-1 receptor potency. Surface plasmon resonance revealed that both small molecules were able to confer albumin affinity to GLP-1 and indicated that affinity is increased for divalent analogues. In lean mice, the divalent GLP-1 analogues were superior to monovalent analogues with respect to control of glucose homeostasis and suppression of food intake. Importantly, divalent GLP-1 analogues showed efficacy comparable to liraglutide, an antidiabetic GLP-1 analogue that carries a long-chain fatty acid. Finally, pharmacokinetic investigations of a divalent GLP-1 analogue demonstrated a promising gain in circulatory half-life and absorption time compared to its monovalent equivalent.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

Chemoselective Reactions for the Synthesis of Glycoconjugates from Unprotected Carbohydrates.

Glycobiology is the comprehensive biological investigation of carbohydrates. The study of the role and function of complex carbohydrates often requires the attachment of carbohydrates to surfaces, their tagging with fluorophores, or their conversion into natural or non-natural glycoconjugates, such as glycopeptides or glycolipids. Glycobiology and its "omics", glycomics, require easy and robust chemical methods for the construction of these glycoconjugates. This review gives an overview of the rapidly expanding field of chemical reactions that selectively convert unprotected carbohydrates into glycoconjugates through the anomeric position. The discussion is divided in terms of the anomeric bond type of the newly formed glycoconjugates, including O-, N-, S-, and C-glycosides.

Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

Electrochemical detection of glutathione S-transferase: an important enzyme in the cell protective mechanism against oxidative stress.

Oxidative stress arises when the antioxidant capacity of cells to clean the excess production of reactive oxygen species (ROS) decreases. Several human diseases seem to be related with an increment in the oxidative stress. In this regard, GSH present in the cells works by neutralizing ROS and other xenobiotics through the glutathione S-transferase (GST) enzyme. Thus, the level of expression of GST is an important factor in determining the sensitivity of cells to toxic chemicals or xenobiotic compounds. Therefore, the detection of GST levels is fundamental in the clinical diagnosis of ROS-related diseases. Here, we describe a methodology, based on the voltammetric properties of the ferrocene group (used as electrochemical probe), which can be applied for selective detection of GST levels in human cells. The electrochemical signal measured is associated to the specific interaction of a ferrocenyl-GSH derivate with the G- and H-sites of this enzyme.

ß-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, ß-cyclodextrin, and both simultaneously have been prepared. The interaction of such nanoparticles with ß-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.

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.

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.

Binding properties of ferrocene-glutathione conjugates as inhibitors and sensors for glutathione S-transferases.

The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.