Manuel C. Martos-Maldonado

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.

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.

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.

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 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.

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.His-tagged proteins can undergo N-terminal acylation as an undesired side-reaction. Here, the authors utilize this to develop a method for highly selective acylation and further modification of peptides and proteins using an optimized His sequence and 4-methoxyphenyl esters as acyl donors.