A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds.

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 ß-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been heterologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)-beechwood xylan at 70 °C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabinoxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize ß-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological applications.

Pharmacological Chaperones for GCase that Switch Conformation with pH Enhance Enzyme Levels in Gaucher Animal Models.

Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X-ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH-switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential.

De Novo Design of Selective Quadruplex-Duplex Junction Ligands and Structural Characterisation of Their Binding Mode: Targeting the G4 Hot-Spot.

Targeting the interface between DNA quadruplex and duplex regions by small molecules holds significant promise in both therapeutics and nanotechnology. Herein, a new pharmacophore is reported, which selectively binds with high affinity to quadruplex-duplex junctions, while presenting a poorer affinity for G-quadruplex or duplex DNA alone. Ligands complying with the reported pharmacophore exhibit a significant affinity and selectivity for quadruplex-duplex junctions, including the one observed in the HIV-1 LTR-III sequence. The structure of the complex between a quadruplex-duplex junction with a ligand of this family has been determined by NMR methods. According to these data, the remarkable selectivity of this structural motif for quadruplex-duplex junctions is achieved through an unprecedented interaction mode so far unexploited in medicinal and biological chemistry: the insertion of a benzylic ammonium moiety into the centre of the partially exposed G-tetrad at the interface with the duplex. Further decoration of the described scaffolds with additional fragments opens up the road to the development of selective ligands for G-quadruplex-forming regions of the genome.

De Novo Design of Selective Quadruplex-Duplex Junction Ligands and Structural Characterisation of Their Binding Mode: Targeting the G4 Hot-Spot.

Invited for the cover of this issue are Andrés G. Santana, Carlos González, Juan Luis Asensio and co-workers at Instituto de Química Orgánica General, Instituto de Química-Física Rocasolano and Universidad de La Rioja. The image depicts drug selectivity using a metaphor of an arrow hitting a target. Read the full text of the article at 10.1002/chem.202005026.

Single-Step Glycosylations with 13 C-Labelled Sulfoxide Donors: A Low-Temperature NMR Cartography of the Distinguishing Mechanistic Intermediates.

Glycosyl sulfoxides have gained recognition in the total synthesis of complex oligosaccharides and as model substrates for dissecting the mechanisms involved. Reactions of these donors are usually performed under pre-activation conditions, but an experimentally more convenient single-step protocol has also been reported, whereby activation is performed in the presence of the acceptor alcohol; yet, the nature and prevalence of the reaction intermediates formed in this more complex scenario have comparatively received minimal attention. Herein, a systematic NMR-based study employing both 13 C-labelled and unlabelled glycosyl sulfoxide donors for the detection and monitoring of marginally populated intermediates is reported. The results conclusively show that glycosyl triflates play a key role in these glycosylations despite the presence of the acceptor alcohol. Importantly, the formation of covalent donor/acceptor sulfonium adducts was identified as the main competing reaction, and thus a non-productive consumption of the acceptor that could limit the reaction yield was revealed.

Intramolecular Metal-Free C(sp3)-H Activation Enables a Selective Mono O-Debenzylation of Fully Protected Aminosugars.

Carbamate-bearing benzylated aminosugars undergo an I2/I(III)-promoted intramolecular hydrogen atom transfer (IHAT) followed by a nucleophilic attack to provide polycyclic structures. Thus, suitably positioned benzyl ethers are surgically oxidized into the corresponding mixed N/O-benzylidene acetals, which can be conveniently deprotected under mild acidic conditions to grant access to selectively O-deprotected aminosugars amenable for further derivatization. The scope of this strategy has been proven with a series of furanosic and pyranosic scaffolds. Preliminary mechanistic studies, including Hammett LFER and KIE analyses, support a reaction pathway with nucleophilic cyclization as the rate-determining step.

Dissecting the Essential Role of Anomeric ß-Triflates in Glycosylation Reactions.

Glycosylations promoted by triflate-generating reagents are widespread synthetic methods for the construction of glycosidic scaffolds and glycoconjugates of biological and chemical interest. These processes are thought to proceed with the participation of a plethora of activated high energy intermediates such as the α- and ß-glycosyl triflates, or even increasingly unstable glycosyl oxocarbenium-like species, among which only α-glycosyl triflates have been well characterized under representative reaction conditions. Interestingly, the remaining less accessible intermediates, yet to be experimentally described, seem to be particularly relevant in α-selective processes, involving weak acceptors. Herein, we report a detailed analysis of several paradigmatic and illustrative examples of such reactions, employing a combination of chemical, NMR, kinetic and theoretical approaches, culminating in the unprecedented detection and quantification of the true ß-glycosyl triflate intermediates within activated donor mixtures. This achievement was further employed as a stepping-stone for the characterization of the triflate anomerization dynamics, which along with the acceptor substitutions, govern the stereochemical outcome of the reaction. The obtained data conclusively show that, even for highly dissociative reactions involving ß-close ion pair (ß-CIP) species, the formation of the α-glycoside is necessarily preceded by a bimolecular α → ß triflate interconversion, which under certain circumstances becomes the rate-limiting step. Overall, our results rule out the prevalence of the Curtin-Hammett fast-exchange assumption for most glycosylations and highlight the distinct reactivity properties of α- and ß-glycosyl triflates against neutral and anionic acceptors.

Impact of Aromatic Stacking on Glycoside Reactivity: Balancing CH/π and Cation/π Interactions for the Stabilization of Glycosyl-Oxocarbenium Ions.

Carbohydrate/aromatic stacking represents a recurring key motif for the molecular recognition of glycosides, either by protein binding domains, enzymes, or synthetic receptors. Interestingly, it has been proposed that aromatic residues might also assist in the formation/cleavage of glycosidic bonds by stabilizing positively charged oxocarbenium-like intermediates/transition states through cation/π interactions. While the significance of aromatic stacking on glycoside recognition is well stablished, its impact on the reactivity of glycosyl donors is yet to be explored. Herein, we report the first experimental study on this relevant topic. Our strategy is based on the design, synthesis, and reactivity evaluation of a large number of model systems, comprising a wide range of glycosidic donor/aromatic complexes. Different stacking geometries and dynamic features, anomeric leaving groups, sugar configurations, and reaction conditions have been explicitly considered. The obtained results underline the opposing influence exerted by van der Waals and Coulombic forces on the reactivity of the carbohydrate/aromatic complex: depending on the outcome of this balance, aromatic platforms can indeed exert a variety of effects, stretching from reaction inhibition all the way to rate enhancements. Although aromatic/glycosyl cation contacts are highly dynamic, the conclusions of our study suggest that aromatic assistance to glycosylation processes must indeed be feasible, with far reaching implications for enzyme engineering and organocatalysis.

Synthesis of Chiral Polyhydroxylated Benzimidazoles by a Tandem Radical Fragmentation/Cyclization Reaction: A Straight Avenue to Fused Aromatic-Carbohydrate Hybrids.

The synthesis of benzimidazole-fused iminosugars through a tandem ß-fragmentation-intramolecular cyclization reaction is described. The use of the benzimidazole ring as the internal nucleophile and the use of phenyliodosophthalate (PhI(Phth)), a new metal-free and low toxic hypervalent iodine reagent, are the most remarkable novelties of this synthetic strategy. With this approach, we have demonstrated the usefulness of the fragmentation of anomeric alkoxyl radicals promoted by the PhI(Phth)/I2 system for the preparation of new compounds with potential interest for both medicinal and synthetic chemists.

Facile Formation of ß-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

Overcoming Aminoglycoside Enzymatic Resistance: Design of Novel Antibiotics and Inhibitors.

Resistance to aminoglycoside antibiotics has had a profound impact on clinical practice. Despite their powerful bactericidal activity, aminoglycosides were one of the first groups of antibiotics to meet the challenge of resistance. The most prevalent source of clinically relevant resistance against these therapeutics is conferred by the enzymatic modification of the antibiotic. Therefore, a deeper knowledge of the aminoglycoside-modifying enzymes and their interactions with the antibiotics and solvent is of paramount importance in order to facilitate the design of more effective and potent inhibitors and/or novel semisynthetic aminoglycosides that are not susceptible to modifying enzymes.

Fungal Glycolipid Hydrolase Inhibitors and Their Effect on Cryptococcus neoformans.

Pathogenic fungi kill an estimated 1.3 million people each year. This number is predicted to rise as drug resistance spreads, thus antifungal drugs with novel modes of action are urgently required. Fungal endoglycoceramidase-related proteins 1 and 2 (EGCrP-1 and -2), which hydrolyse glucosylceramide and ergosteryl ß-glucoside, respectively, are important for fungal cell growth and have been identified as potential targets for drug development. A library of iminosugar derivatives was screened against EGCrP-1 and -2, and a number of competitive inhibitors with nanomolar affinities were identified. In addition, a mechanism-based inhibitor was shown to form a covalent derivative with EGCrP-2. Nine of the inhibitors were evaluated against Cryptococcus neoformans. Several showed growth inhibitory activity, but only against a C. neoformans strain lacking the outer fungal polysaccharide capsule; this implies that penetration into the cell is a significant handicap for these inhibitors. Pro-drug versions of these inhibitors could address this issue.

Finding the Right Candidate for the Right Position: A Fast NMR-Assisted Combinatorial Method for Optimizing Nucleic Acids Binders.

Development of strong and selective binders from promiscuous lead compounds represents one of the most expensive and time-consuming tasks in drug discovery. We herein present a novel fragment-based combinatorial strategy for the optimization of multivalent polyamine scaffolds as DNA/RNA ligands. Our protocol provides a quick access to a large variety of regioisomer libraries that can be tested for selective recognition by combining microdialysis assays with simple isotope labeling and NMR experiments. To illustrate our approach, 20 small libraries comprising 100 novel kanamycin-B derivatives have been prepared and evaluated for selective binding to the ribosomal decoding A-Site sequence. Contrary to the common view of NMR as a low-throughput technique, we demonstrate that our NMR methodology represents a valuable alternative for the detection and quantification of complex mixtures, even integrated by highly similar or structurally related derivatives, a common situation in the context of a lead optimization process. Furthermore, this study provides valuable clues about the structural requirements for selective A-site recognition.

Selective modification of the 3''-amino group of kanamycin prevents significant loss of activity in resistant bacterial strains.

Aminoglycosides are highly potent, wide-spectrum bactericidals. N-1 modification of aminoglycosides has thus far been the best approach to regain bactericidal efficiency of this class of antibiotics against resistant bacterial strains. In the present study we have evaluated the effect that both, the number of modifications and their distribution on the aminoglycoside amino groups (N-1, N-3, N-6' and N-3''), have on the antibiotic activity. The modification of N-3'' in the antibiotic kanamycin A is the key towards the design of new aminoglycoside antibiotics. This derivative maintains the antibiotic activity against aminoglycoside acetyl-transferase- and nucleotidyl-transferase-expressing strains, which are two of the most prevalent modifying enzymes found in aminoglycoside resistant bacteria.

Illuminating a Solvent-Dependent Hierarchy for Aromatic CH/π Complexes with Dynamic Covalent Glyco-Balances.

CH/π interactions are prevalent among aromatic complexes and represent invaluable tools for stabilizing well-defined molecular architectures. Their energy contributions are exceptionally sensitive to various structural and environmental factors, resulting in a context-dependent nature that has led to conflicting findings in the scientific literature. Consequently, a universally accepted hierarchy for aromatic CH/π interactions has remained elusive. Herein, we present a comprehensive experimental investigation of aromatic CH/π complexes, employing a novel approach that involves isotopically labeled glyco-balances generated in situ. This innovative strategy not only allows us to uncover thermodynamic insights but also delves into the often less-accessible domain of kinetic information. Our analyses have yielded more than 180 new free energy values while considering key factors such as solvent properties, the interaction geometry, and the presence and nature of accompanying counterions. Remarkably, the obtained results challenge conventional wisdom regarding the stability order of common aromatic complexes. While it was believed that cationic CH/π interactions held the highest strength, followed by polarized CH/π, nonpolarized CH/π, and finally anionic CH/π interactions, our study reveals that this hierarchy can be subverted depending on the environment. Indeed, the performance of polarized CH/π interactions can match or even outcompete that of cationic CH/π interactions making them a more reliable stabilization strategy across the entire spectrum of solvent polarity. Overall, our results provide valuable guidelines for the selection of optimal interacting partners in every chemical environment, allowing the design of tailored aromatic complexes with applications in supramolecular chemistry, organocatalysis, and/or material sciences.

A dynamic combinatorial approach for the analysis of weak carbohydrate/aromatic complexes: dissecting facial selectivity in CH/π stacking interactions.

A dynamical combinatorial approach for the study of weak carbohydrate/aromatic interactions is presented. This methodology has been employed to dissect the subtle structure-stability relationships that govern facial selectivity in these supramolecular complexes.

Synthesis of branched iminosugars through a hypervalent iodine(III)-mediated radical-polar crossover reaction.

The synthesis of a novel type of branched iminosugars is described. This synthetic strategy is based on two key reactions: first, an aldol reaction with formaldehyde in order to introduce selectively the hydroxymethyl branch, and second, a tandem ß-fragmentation-intramolecular cyclization reaction. The combination of both reactions afforded a battery of compounds exhibiting a great structural complexity, with the concomitant formation of a quaternary center, starting from readily available aldoses. With this approach we have demonstrated the usefulness of the fragmentation of anomeric alkoxyl radicals (ARF) promoted by the PhIO/I2 system for the preparation of new compounds with potential interest for both medicinal and synthetic chemists.

Multiple keys for a single lock: the unusual structural plasticity of the nucleotidyltransferase (4')/kanamycin complex.

The most common mode of bacterial resistance to aminoglycoside antibiotics is the enzyme-catalysed chemical modification of the drug. Over the last two decades, significant efforts in medicinal chemistry have been focused on the design of non- inactivable antibiotics. Unfortunately, this strategy has met with limited success on account of the remarkably wide substrate specificity of aminoglycoside-modifying enzymes. To understand the mechanisms behind substrate promiscuity, we have performed a comprehensive experimental and theoretical analysis of the molecular-recognition processes that lead to antibiotic inactivation by Staphylococcus aureus nucleotidyltransferase 4'(ANT(4')), a clinically relevant protein. According to our results, the ability of this enzyme to inactivate structurally diverse polycationic molecules relies on three specific features of the catalytic region. First, the dominant role of electrostatics in aminoglycoside recognition, in combination with the significant extension of the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The motion deduced for the bound antibiotic seem to be essential for the enzyme action and probably provide a mechanism to explore alternative drug inactivation modes. Second, the nucleotide recognition is exclusively mediated by the inorganic fragment. In fact, even inorganic triphosphate can be employed as a substrate. Third, ANT(4') seems to be equipped with a duplicated basic catalyst that is able to promote drug inactivation through different reactive geometries. This particular combination of features explains the enzyme versatility and renders the design of non-inactivable derivatives a challenging task.

Binding-driven reactivity attenuation enables NMR identification of selective drug candidates for nucleic acid targets.

NMR methods, and in particular ligand-based approaches, are among the most robust and reliable alternatives for binding detection and consequently, they have become highly popular in the context of hit identification and drug discovery. However, when dealing with DNA/RNA targets, these techniques face limitations that have precluded widespread application in medicinal chemistry. In order to expand the arsenal of spectroscopic tools for binding detection and to overcome the existing difficulties, herein we explore the scope and limitations of a strategy that makes use of a binding indicator previously unexploited by NMR: the perturbation of the ligand reactivity caused by complex formation. The obtained results indicate that ligand reactivity can be utilised to reveal association processes and identify the best binders within mixtures of significant complexity, providing a conceptually different reactivity-based alternative within NMR screening methods.