Unlocking the Hydrolytic Mechanism of GH92 α-1,2-Mannosidases: Computation Inspires the use of C-Glycosides as Michaelis Complex Mimics.

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

Silicon-bridged (1→1)-disaccharides: an umpoled glycomimetic scaffold.

Modification of the carbohydrate scaffold is an important theme in drug and vaccine discovery. Therefore, the preparation of novel types of glycomimetics is of interest in synthetic carbohydrate chemistry. In this manuscript, we present an early investigation of the synthesis, structure, and conformational behaviour of (1→1)-Si-disaccharides as a novel type of glycomimetics arising from the replacement of interglycosidic oxygen with a dimethyl-, methylpropyl-, or diisopropylsilyl linkage. We accomplished the preparation of this unusual group of umpoled compounds by the reaction of lithiated glycal or 2-oxyglycal units with dialkyldichlorosilanes. We demonstrated the good stability of the "Si-glycosidic" linkage under acidic conditions even at elevated temperatures. Next, we described the conformational landscape of these compounds by the combination of in silico modelling with spectroscopic and crystallographic methods. Finally, we explained the observed conformational flexibility of these compounds by the absence of gauche stabilizing effects that are typically at play in natural carbohydrates.

Facile Approach to C-Glucosides by Using a Protecting-Group-Free Hiyama Cross-Coupling Reaction: High-Yielding Dapagliflozin Synthesis.

Invited for the cover of this issue is Kamil Parkan and co-workers at University of Chemistry and Technology and Institute of Organic Chemistry and Biochemistry, Prague. The cover graphic depicts a schematic representation of the assembly of aryl-C-glycosides based on a protecting-group-free Hiyama reaction. Read the full text of the article at 10.1002/chem.202101052.

Facile Approach to C-Glucosides by Using a Protecting-Group-Free Hiyama Cross-Coupling Reaction: High-Yielding Dapagliflozin Synthesis.

Access to unprotected (hetero)aryl pseudo-C-glucosides via a mild Pd-catalysed Hiyama cross-coupling reaction of protecting-group-free 1-diisopropylsilyl-d-glucal with various (hetero)aryl halides has been developed. In addition, selected unprotected pseudo-C-glucosides were stereoselectively converted into the corresponding α- and ß-C-glucosides, as well as 2-deoxy-ß-C-glucosides. This methodology was applied to the efficient and high-yielding synthesis of dapagliflozin, a medicament used to treat type 2 diabetes mellitus. Finally, the versatility of our methodology was proved by the synthesis of other analogues of dapagliflozin.

Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed ß-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed ß-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Polyvalent C-glycomimetics based on l-fucose or d-mannose as potent DC-SIGN antagonists.

The C-type lectin DC-SIGN expressed on immature dendritic cells is a promising target for antiviral drug development. Previously, we have demonstrated that mono- and divalent C-glycosides based on d-manno and l-fuco configurations are promising DC-SIGN ligands. Here, we described the convergent synthesis of C-glycoside dendrimers decorated with 4, 6, 9, and 12 α-l-fucopyranosyl units and with 9 and 12 α-d-mannopyranosyl units. Their affinity against DC-SIGN was assessed by surface plasmon resonance (SPR) assays. For comparison, parent O-glycosidic dendrimers were synthesized and tested, as well. A clear increase of both affinity and multivalency effect was observed for C-glycomimetics of both types (mannose and fucose). However, when dodecavalent C-glycosidic dendrimers were compared, there was no difference in affinity regarding the sugar unit (l-fuco, IC50 17 µM; d-manno, IC50 12 µM). For the rest of glycodendrimers with l-fucose or d-mannose attached by the O- or C-glycosidic linkage, C-glycosidic dendrimers were significantly more active. These results show that in addition to the expected physiological stability, the biological activity of C-glycoside mimetics is higher in comparison to the corresponding O-glycosides and therefore these glycomimetic multivalent systems represent potentially promising candidates for targeting DC-SIGN.

Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of HIV-1 structural protein assembly.

In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC50 values higher than 28µM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC50 values ranging from 3 to 60µM. The congeners with a fluoro substituent introduced at the 4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding to the C-terminal domain of the capsid protein.

Modular Stereoselective Synthesis of (1→2)-C-Glycosides based on the sp(2) -sp(3) Suzuki-Miyaura Reaction.

This work reports a modular and rapid approach to the stereoselective synthesis of a variety of α- and ß-(1→2)-linked C-disaccharides. The key step is a Ni-catalyzed cross-coupling reaction of D-glucal pinacol boronate with alkyl halide glycoside easily prepared from commercially available D-glucal. The products of this sp(2) -sp(3) cross-coupling reaction can be converted to glucopyranosyl, mannopyranosyl, or 2-deoxy-glucopyranosyl C-mannopyranosides by one- or two-step stereoselective oxidative-reductive transformations. To the best of our knowledge, we demonstrated the first synthetic application of a challenging sp(2) -sp(3) Suzuki-Miyaura cross-coupling reaction in carbohydrate chemistry.

Cross-metathesis reaction of α- and ß-vinyl C-glycosides with alkenes.

Cross-metathesis of α- and ß-vinyl C-deoxyribosides and α-vinyl C-galactoside with various terminal alkenes under different conditions was studied. The cross-metathesis of the former proceeded with good yields of the corresponding products in ClCH2CH2Cl the latter required the presence of CuI in CH2Cl2 to achieve good yields of the products. A simple method for the preparation of α- and ß-vinyl C-deoxyribosides was also developed. In addition, feasibility of deprotection and further transformations were briefly explored.

Cross-coupling reaction of saccharide-based alkenyl boronic acids with aryl halides: the synthesis of bergenin.

A convenient synthetic pathway enabling D-glucal and D-galactal pinacol boronates to be prepared in good isolated yields was achieved. Both pinacol boronates were tested in a series of cross-coupling reactions under Suzuki-Miyaura cross-coupling conditions to obtain the corresponding aryl, heteroaryl, and alkenyl derivatives in high isolated yields. This methodology was applied to the formal synthesis of the glucopyranoside moiety of papulacandin D and the first total synthesis of bergenin.

On the origin of the electronic and magnetic circular dichroism of naphthyl C-glycosides: Anomeric configuration.

Aryl C-glycosides, in which the glycosidic bond is changed to a carbon-carbon bond, are an important family of biologically-active compounds. They often serve as secondary metabolites or exhibit antibiotic and cytostatic activities. Their stability to hydrolysis has made them attractive targets for new drugs. Their conformational behavior often strongly influences the resulting function. Their detailed structural and conformational description is thus highly desirable. This work studies the structure of three different naphthyl C-glycosides using UV-vis absorption as well as electronic and magnetic circular dichroism. It also describes their conformational preferences using a combination of molecular dynamics and DFT calculations. The reliability of these preferences has been verified by simulations of spectral properties and a comparison with their measured spectra. In particular, ECD spectroscopy has been shown to distinguish easily between α- and ß-pseudoanomers of aryl C-glycosides. Computer simulations and spectral decomposition have revealed how the resulting ECD patterns of the naphthyl glycosides studied are influenced by different conformer populations. In conclusion, reliable ECD patterns cannot be calculated by separating the naphthyl rotation from other conformational motions. MCD patterns have been similar for all the naphthyl C-glycosides studied. No clear diagnostic features have been found for either the pseudoanomeric configuration or the preferred hydroxymethyl rotamer. Nevertheless, the work has demonstrated the potential of MCD for the study of aryl glycosides interacting with proteins.

The synthesis and characterization of electron-poor glycosylamines and derived glycosylamides.

This paper describes a unified approach toward diglycosylamines using methanolic ammonia. All the glycosylamines prepared have been fully characterized, and their anomeric configuration has been determined. The article presents a novel method for the N-acylation of diglycosylamines and other electron-poor glycosylamines, which employs nitromethane as a solvent in carboxylic anhydride acylation under acidic conditions. The feasibility of this transformation is represented by a wide range of reaction substrates. All glycosylamides are formed solely with ß-configuration. These two reactions constitute a simple and effective route to the synthesis of a novel class of compounds with an N-glycosidic linkage.

Selection of Galectin-Binding Ligands from Synthetic Glycopeptide Libraries.

Galectins, a class of carbohydrate-binding proteins, play a crucial role in various physiological and disease processes. Therefore, the identification of ligands that efficiently bind these proteins could potentially lead to the development of new therapeutic compounds. In this study, we present a method that involves screening synthetic click glycopeptide libraries to identify lectin-binding ligands with low micromolar affinity. Our methodology, initially optimized using Concanavalin A, was subsequently applied to identify binders for the therapeutically relevant galectin 1. Binding affinities were assessed using various methods and showed that the selected glycopeptides exhibited enhanced binding potency to the target lectins compared to the starting sugar moieties. This approach offers an alternative means of discovering galectin-binding ligands as well as other carbohydrate-binding proteins, which are considered important therapeutic targets.

Covalent stabilization: a sturdy molecular square from reversible metal-ion-directed self-assembly.

Supramolecular self-assembly using weak interactions under quasi-equilibrium conditions has provided easy access to very complex but often quite fragile molecules. We now show how a labile structure obtained from reversible transition-metal-directed self-assembly of rods and connectors serves as a template that can be converted into a sturdy structure of identical topology and similar geometry. The process consists of Cu(I)-catalyzed replacement of all rods or connectors terminated with pyridines for analogues terminated with ethynyls, converting dative N→Pt(+) bonds into covalent C-Pt bonds. The procedure combines the facility and high yield of reversible self-assembly with the robustness of covalent synthesis.

Straightforward synthesis of protected 2-hydroxyglycals by chlorination-dehydrochlorination of carbohydrate hemiacetals.

A straightforward and scalable method for the synthesis of protected 2-hydroxyglycals is described. The approach is based on the chlorination of carbohydrate-derived hemiacetals, followed by an elimination reaction to establish the glycal moiety. 1,2-dehydrochlorination reactions were studied on a range of glycosyl chlorides to provide suitable reaction conditions for this transformation. Benzyl ether, isopropylidene and benzylidene protecting groups, as well as interglycosidic linkage, were found to be compatible with this protocol. The described method is operationally simple and allows for the quick preparation of 2-hydroxyglycals with other than ester protecting groups, providing a feasible alternative to existing methods.

Fucosylated inhibitors of recently identified bangle lectin from Photorhabdus asymbiotica.

A recently described bangle lectin (PHL) from the bacterium Photorhabdus asymbiotica was identified as a mainly fucose-binding protein that could play an important role in the host-pathogen interaction and in the modulation of host immune response. Structural studies showed that PHL is a homo-dimer that contains up to seven L-fucose-specific binding sites per monomer. For these reasons, potential ligands of the PHL lectin: α-L-fucopyranosyl-containing mono-, di-, tetra-, hexa- and dodecavalent ligands were tested. Two types of polyvalent structures were investigated - calix[4]arenes and dendrimers. The shared feature of all these structures was a C-glycosidic bond instead of the more common but physiologically unstable O-glycosidic bond. The inhibition potential of the tested structures was assessed using different techniques - hemagglutination, surface plasmon resonance, isothermal titration calorimetry, and cell cross-linking. All the ligands proved to be better than free L-fucose. The most active hexavalent dendrimer exhibited affinity three orders of magnitude higher than that of standard L-fucose. To determine the binding mode of some ligands, crystal complex PHL/fucosides 2 - 4 were prepared and studied using X-ray crystallography. The electron density in complexes proved the presence of the compounds in 6 out of 7 fucose-binding sites.

MOP and EE Protecting Groups in Synthesis of α- or ß-Naphthyl-C-Glycosides from Glycals.

The development of effective protection strategies is essential in the synthesis of complex carbohydrates and glycomimetics. This article describes a versatile four-stage protocol for the synthesis of α- or ß-aryl-C-glycosides from unprotected d-glycals using two acetal protecting groups, ethoxyethyl and methoxypropyl, which are stable under harsh basic conditions and convenient for the C-1 metalation of glycals. Their stability was investigated in subsequent cross-coupling reactions with 1-iodonaphthalene followed by oxidative/reductive transformations to naphthyl-C-glycosides.

Limitations in the description of conformational preferences of C-disaccharides: The (1 → 3)-C-mannobiose case.

Conformational preferences of two C-glycosyl analogues of Manp-(1 â†’ 3)-Manp, were studied using a combined method of theoretical and experimental chemistry. Molecular dynamics was utilized to provide conformational behavior along C-glycosidic bonds of methyl 3-deoxy-3-C-[(α-d-mannopyranosyl)methyl]-α-d- and l-mannopyranosides. The OPLS2005 and Glycam06 force fields were used. Simulations were performed with explicit water (TIP3P) and methanol. Results were compared with a complete conformational scan at the MM4 level with the dielectric constant corresponding to methanol. In order to verify predicted conformational preferences, vicinal 3JHH NMR coupling constants were calculated by the Karplus equation on simulated potential energy surfaces (PES). A set of new parameters for the Karplus equation was also designed. Predicted 3JHH were compared with experimental data. We also used reverse methodology, in which the 3JHH coupling constants were calculated at the DFT level for each family of (ϕ, ψ)-conformers separately and then experimental values were decomposed onto calculated 3JHH couplings in order to obtain experimentally derived populations of conformers. As an alternative method of evaluation of preferred conformers, analysis of sensitive 13C chemical shifts was introduced. We were able to thoroughly discuss several fundamental issues in predictions of preferred conformers of C-saccharides, such as the solvent effect, reliability of the force field, character of empirical Karplus equation or applicability of NMR parameters in predictions of conformational preferences in general.

Nonhydrolyzable C-disaccharides, a new class of DC-SIGN ligands.

The discovery of effective ligands for DC-SIGN receptor is one of the most challenging concepts of antiviral drug design due to the importance of this C-type lectin in infection processes. DC-SIGN recognizes mannosylated and fucosylated oligosaccharides but glycosidic linkages are accessible to both chemical and enzymatic degradations. To avoid this problem, the synthesis of stable glycoside mimetics has attracted increasing attention. In this work we establish for the first time mono- and divalent C-glycosides based on d-manno and l-fuco configurations as prospective DC-SIGN ligands. In particular, the l-fucose glycomimetics were more active than the respective d-mannose ones. The highest affinity was assessed for simple 1,4-bis(α-l-fucopyranosyl)butane (SPR: IC50 0.43 mM) that displayed about twice higher activity than natural ligand Lex. Our results make C-glycosides attractive candidates for multivalent presentations.

Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations.