Fernando Hernandez-Mateo

Azide–Alkyne 1,3-Dipolar Cycloadditions: a Valuable Tool in Carbohydrate Chemistry

Recent applications of the 1,3-dipolar cycloaddition reaction of azides and alkynes in carbohydrate chemistry are summarized in the present review. The efficient catalyzed version of this reaction, also referred to as one of the so-called click chemistry reactions, has joined in a short period of time a select group of the most efficient and useful organic reactions. In particular, its application in the carbohydrate field has shown its value as a synthetic tool allowing the preparation of a wide range of carbohydrate-containing molecular constructs such as glycopeptides, glycoaminoacids, glycoclusters, glycodendrimers, glycopolymers, glycosylated biomolecules, and immobilization of carbohydrates onto solid surfaces (glycoarrays) as the most representative examples. In the majority of cases, the formed 1,2,3-triazole heterocycle plays the role of a linking tether between the coupling partners.

Vinyl sulfone bifunctional tag reagents for single-point modification of proteins.

The introduction of multiple labels onto biomolecules is a challenge. We report herein the synthesis of vinyl sulfone derivatized bifunctional tag single-attachment-point reagents (BTSAP) bearing biotin and a fluorescent tag and their applications in proteins for the introduction of multiple labels by means of the Michael-type addition of the electrophilic vinyl sulfone group. These BTSAP reagents were easily synthesized by a two-step chemical strategy involving the preparation of alkyne vinyl sulfone derivatized tags (AVST) and subsequent click CuAAC attachment of a second azide functionalized tag. The direct coupling of BTSAP reagents with the low reactive protein horseradish peroxidase (HRP) turned it into a dual reporter group (i.e., fluorescence and peroxidase activity) that may be coupled to any recognition system via biotin-avidin affinity. The AVST compounds are not mere synthetic intermediates for the preparation of BTSAP reagents but valuable clickable self-reporting compounds that allow the simultaneous introduction in proteins of an alkyne function and labeling when conjugated via the vinyl sulfone group. The implementation of these clickable AVST compounds in a CuAAC-based sequential approach also allows attainment of the dual labeling of HRP. This approach yields equivalent results in terms of fluorescent labeling, specific activity, and functionality of the biotin tag when compared with the direct bifunctional labeling by the BTSAP reagent. However, for life science this direct approach is more convenient since it avoids the use of copper catalysis, overcoming the toxicity drawback of this metal in biological systems.

SCOPE AND APPLICATIONS OF ACTIVE AND LATENT THIOGLYCOSYL DONORS. PART 4

Abstract The relative reactivity of various thioglycosyl donors having ethyl, phenyl, or para-substituted phenyl groups with electron donating (N-Ac) or electron withdrawing (NO2) substituents were compared using 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose (3) as standard glycosyl acceptor. The reactivity order was found to decrease from ethyl > phenyl > p-acetamidophenyl > p-nitrophenyl. In the latter situation, when the thioglycosyl donor was also equipped with “disarming” ester protecting groups, they were found to be inert or inactive toward common thiophilic promotors. Alternatively, it was possible to selectively activate the “armed” perbenzylated p-nitrophenyl 1-thio-β-D-galactopyranoside (21) in the presence of the corresponding “disarmed” perbenzoylated p-nitrophenyl 2,3,4-tri-O-benzoyl-1-thio-β-D-galactopyranoside (15) which served as the glycosyl acceptor. When both “armed” perbenzylated thioglycosides 7 and 25 were used as thioglycosyl donor and thioglycosyl acceptor, respectively, the mil...

Vinyl sulfone functionalized silica: a “ready to use” pre-activated material for immobilization of biomolecules

The combination of silica as support and vinyl sulfone as reactive group led to a pre-activated material that readily reacts to form covalent bonds by Michael-type addition with both amine and thiol groups naturally occurring in biomolecules in mild conditions compatible with the biological nature of the enzymes. A simple two step synthetic strategy was designed to access this functionalized hybrid material. Two types of vinyl sulfone silicas (N-type and S-type) differing in the chemical nature of the linkers between the silica particle and the reactive vinyl sulfone group were prepared by implementation of this strategy. The capabilities of those vinyl sulfone silicas were evaluated with the model enzymes invertase, lactase and lysozyme. Both S-type and N-type vinyl sulfone silicas coupled efficiently with the model enzymes even at 4 °C by simple combination of the species and the immobilized enzymes retained the enzymatic activity. The linker showed to play a major role in the non covalent interactions between the enzymes and the silicas. In terms of capacity, the S-type material is the best option although its poor flow rate when packed in columns invalidates its applications for low pressure liquid chromatography. The capabilities of the N-type material were successfully put to the test as a pre-packed column for the immobilization of invertase and further demonstrated with two real cases of relevance in proteomics: (i) purification of glutathione-S-transferase (GST) and (ii) identification of proteins that interact with thioredoxin h2 from Pisum sativum.

Vinyl Sulfone Functionalization: A Feasible Approach for the Study of the Lectin! Carbohydrate Interactions

Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).

Divinyl Sulfone Cross-Linked Cyclodextrin-Based Polymeric Materials: Synthesis and Applications as Sorbents and Encapsulating Agents

The aim of this study was to evaluate the crosslinking abilities of divinyl sulfone (DVS) for the preparation of novel water-insoluble cyclodextrin-based polymers (CDPs) capable of forming inclusion complexes with different guest molecules. Reaction of DVS with native α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and/or starch generates a variety of homo- and hetero-CDPs with different degrees of crosslinking as a function of the reactants’ stoichiometric ratio. The novel materials were characterized by powder X-ray diffraction, electron microscopy and for their sorption of phenol and 4-nitrophenol. They were further evaluated as sorbents with phenolic pollutants (bisphenol A and β-naphthol) and bioactive compounds (the hormone progesterone and curcumin). Data obtained from the inclusion experiments show that the degree of cross-linking has a minor influence on the yield of inclusion complex formation and highlight the important role of the CDs, supporting a sorption process based on the formation of inclusion complexes. In general, the inclusion processes are better described by a Freundlich isotherm although an important number of them can also be fitted to the Langmuir isotherm with R2 ≥ 0.9, suggesting a sorption onto a monolayer of homogeneous sites.

Synthesis and transformations of 2-deoxy-2-iodo-pyranosyl isothiocyanates from glycals

Abstract A convenient one-step synthesis of 2-deoxy-2-iodoglycosyl isothiocyanates from monosaccharidic and disaccharidic glycals is reported. Treatment of iodoisothiocyanates 7, 11, 13–15 with ammonia gives the corresponding 2-amino-thiazolines 16, 17, 23–25 , respectively. Under the same conditions, iodoisothiocyanate 8 affords the iodothiourea 18 . 2-Amino-2-thiazolines 23–25 can be readily transformed into thiazolidin-2-ones 26–28 . Reduction of compounds 7, 8, 13–15 by tributyltin hydride yields the corresponding N-(2′-deoxyglycosyl)thioureas 21, 22, 29–31 .

Polyelectrolyte Complexes of Low Molecular Weight PEI and Citric Acid as Efficient and Nontoxic Vectors for in Vitro and in Vivo Gene Delivery

Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.

Synthetic Applications of Cyclic Sulfites, Sulfates and Sulfamidates in Carbohydrate Chemistry

Cyclic sulfites, sulfates and sulfamidates derived from saccharides have emerged as valuable and versatile synthons for the preparation of a wide variety of modified sugars and sugar related compounds owing to the combination of the easily and efficient forma- tion of these chemical sulfur derivatives from diols and aminoalcohols with the high chemical stability and enhanced reactivity of those functions towards nucleophiles. The state-of-the-art in this area is covered in the present review using the relative position of the diols and the aminoalcohol functions on the saccharide skeleton and the nature of the nucleophilic reagent (oxygenated nucleophiles, halogens, sulphur nucleophiles, nitrogen nucleophiles, and carbon nucleophiles) as expositive criteria. A comprehensive outlook of the applications of these sulfur derivatives in the preparation of compounds such as azido sugars, halo sugars, thiosugars, pseudo-saccharides, radiotrac- ers, enzymatic inhibitors, anticonvulsant agents, surfactants amongst others is also given.

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.