Carba-Sugar Analogs of Glucosamine-6-Phosphate: New Activators for the glmS Riboswitch.

Riboswitches are 5'-untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistance against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self-cleavage upon binding to glucosamine-6-phosphate (GlcN6P). Previously, we showed that carba glucosamine-6-phosphate (carba-GlcN6P) induces self-cleavage of the riboswitch with a potency similar to that of GlcN6P. Here, we report a synthetic approach to a new class of carba-GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis followed by a hydroboration. The strategy gives access to libraries of carba-GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.

A Diversifiable Synthetic Platform for the Discovery of New Carbasugar SGLT2 Inhibitors Using Azide-Alkyne Click Chemistry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are clinically available to control blood glucose levels in diabetic patients via an insulin-independent mechanism. It was found that some carbasugar analogs of known SGLT2 inhibitors exert a high inhibiting ability toward SGLT2 and have a prolonged blood glucose lowering effect. In this study, we designed new candidates of carbasugar SGLT2 inhibitor that can be synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) into an aromatic ring, which is a part of the pharmacophore at the final stage in the synthetic protocol for the easier discovery of superior SGLT2 inhibitors. Based on the results of molecular docking studies, some selected compounds have been synthesized. Evaluation of these compounds using a cell-based assay revealed that the majority of these compounds had SGLT2 inhibitory activity in a dose-dependent manner. The SGLT2 inhibitory activity of 7b and 7c was almost equal to that of SGLT2 inhibitors in current use. Furthermore, molecular dynamics simulations also revealed that 7c is a promising novel SGLT2 inhibitor.

A Synthetic Approach to Several C7-Carbasugars via a Key Intramolecular Morita-Baylis-Hillman Reaction.

A new synthetic strategy for C7-carbasugars is developed via an intramolecular Morita-Baylis-Hillman reaction, in which a substituted dial precursor prepared from d-mannose cyclizes smoothly in the presence of DMAP to afford polyhydroxylated cyclohex-1-enecarbaldehyde with good yield. By employment of the cyclization products as key intermediates, the first syntheses of carbasugar ester 1 and epicorepoxydon A, as well as practical syntheses of epoxydines B and C, (-)-MK7607, (-)-streptol, and (-)-gabosine E are achieved.

Diastereo- and Enantioselective Inverse-Electron-Demand Diels-Alder Cycloaddition between 2-Pyrones and Acyclic Enol Ethers.

A broadly applicable diastereo- and enantioselective inverse-electron-demand Diels-Alder reaction of 2-pyrones and acyclic enol ethers is reported herein. Using a copper(II)-BOX catalytic system, bridged bicyclic lactones are obtained in very high yields (up to 99 % yield) and enantioselectivities (up to 99 % ee) from diversely substituted 2-pyrones and acyclic enol ethers. Mechanistic experiments as well as DFT calculations indicate the occurrence of a stepwise mechanism. The synthetic potential of the bridged bicyclic lactones is showcased by the enantioselective synthesis of polyfunctional cyclohexenes and cyclohexadienes, as well as a carbasugar unit.

Development of Non-Hydrolysable Oligosaccharide Activity-Based Inactivators for Endoglycanases: A Case Study on α-1,6 Mannanases.

There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for α-1,6 endo-mannanases. 3-D structures at 1.35-1.47 Šresolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.

The Role of Fluorine in Glycomimetic Drug Design.

Glycans are well established to play important roles at various stages of infection and disease, and ways to modulate these interactions have been sought as novel therapies. The use of native glycan structures has met with limited success, which can be attributed to their characteristic high polarity (e.g., low binding affinities) and inherently poor pharmacokinetic properties (e.g., short drug-target residence times, rapid renal excretion), leading to the development of 'glycomimetics'. Fluorinated drugs have become increasingly common over recent decades, with fluorinated glycomimetics offering some unique advantages. Deoxyfluorination maintains certain electrostatic interactions, while concomitantly reducing net polarity through 'polar hydrophobicity', improving residence times and binding affinities. Fluorination destabilizes the oxocarbenium transition state associated with metabolic degradation, and can restore exo- and endo-anomeric effects in C-glycosides and carbasugars. Lastly, it has shown great utility in radiotracer development and enhancement of antigenicity in glycan-based vaccines. Owing to synthetic challenges, fluorinated glycomimetics have been somewhat underutilized to date, but methodological improvements will advance their use in glycomimetic drugs.

Conformationally Controlled Reactivity of Carbasugars Uncovers the Choreography of Glycoside Hydrolase Catalysis.

Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some GH families, including α-amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based carbasugars of α-d-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast α-glucosidase. The linear free energy relationships between rate constants and pKa of the leaving group are curved upward, which is indicative of a change in mechanism, with the better leaving groups reacting by an SN1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid SN2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudoglycosidic bond cleavage that occurs via SN1 transition states that include nonproductive binding of the leaving group to the enzyme. In total, we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of the enzyme and natural substrate undergoes which align the nucleophile for efficient catalysis.

Synthesis of Highly Oxygenated Bicyclic Carbasugars. Remarkable Difference in the Reactivity of the d-gluco and d-xylo- Derived Trienes.

2,3,4-Tri-O-benzyl-D-xylopyranose was used as a starting material in the preparation of the corresponding triene, which underwent smooth cyclization to a polyhydroxylated hydrindane, as a single diastereoisomer. The analogous triene prepared from D-glucose did not undergo any cyclization even under high pressure.

Selective synthesis of 3-deoxy-5-hydroxy-1-amino-carbasugars as potential α-glucosidase inhibitors.

A convenient synthesis of novel 3-deoxy-5-hydroxy-1-aminocarbasugars was developed here. The benzyl-protected glucose-derived ketone 12 was selectively converted in high yield to enone 13via retro-Michael elimination of BnOH. The double bond of 13 was regio- and stereo-selectively reduced by the induction of C4-α-OBn to the multi-functionalized 15. 15 contained all the functionalities with similar configurations to carbasugars but with 3-H and 5-OH in the ring, and it would be a very interesting building block for organic synthesis or for bioactive compounds. As one application, 15 was further transformed into 1-amino-carbasugars by the reductive amination and final deprotection of benzyls. The targets were subjected to the in vitro inhibitory activity test against sucrase or maltase. The inhibitory activity of 17b, 17h or 17j against sucrase was nearly similar to that of voglibose. In comparison with voglibose, in vivo results similarly showed that 17b, 17h or 17j could lower the post-prandial blood glucose level after sucrose loading in healthy male ICR mice, while miglitol or acarbose was less effective. The molecular modeling study of some targets or voglibose with human sucrase could explain the inhibiting action.

Remarkable Effect of [Li(G4)]TFSI Solvate Ionic Liquid (SIL) on the Regio- and Stereoselective Ring Opening of α-Gluco Carbasugar 1,2-Epoxides.

Carba analogues of biologically relevant natural carbohydrates are promising structures for the development of future drugs endowed with enhanced hydrolytic stability. An open synthetic challenge in this field is the optimization of new methodologies for the stereo- and regioselective opening of α-gluco carbasugar 1,2-epoxides that allow for the preparation of pseudo mono- and disaccharides of great interest. Therefore, we investigated the effect of Lewis acids and solvate ionic liquids (SILs) on the epoxide ring opening of a model substrate. Of particular interest was the complete stereo- and regioselectivity, albeit limited to simple nucleophiles, toward the desired C(1) isomer that was observed using LiClO4. The results obtained with SILs were also remarkable. In particular, Li[NTf2]/tetraglyme ([Li(G4)]TFSI) was able to function as a Lewis acid and to direct the attack of the nucleophile preferentially at the pseudo anomeric position, even with a more complex and synthetically interesting nucleophile. The regioselectivity observed for LiClO4 and [Li(G4)]TFSI was tentatively ascribed to the formation of a bidentate chelating system, which changed the conformational equilibrium and ultimately permitted a trans-diaxial attack on C(1). To the best of our knowledge, we report here the first case in which SILs were successfully employed in a ring-opening process of epoxides.

Regio- and stereospecific synthesis of rac-carbasugar-based cyclohexane pentols; Investigations of their α- and ß-glucosidase inhibitions.

In the present study, (3aR,7aS)-1,3,3a,4,7,7a-hexahydroisobenzofuran was submitted to photooxygenation and two isomeric hydroperoxides were successfully obtained. Without any further purification, reduction of the hydroperoxides with titanium tetraisopropoxide catalyzed by dimethyl sulfide gave two alcohol isomers in high yields. After acetylation of alcohol with Ac2O in pyridine, epoxidation reaction of formed monoacetates with m-CPBA, then chromatographed and followed by hydrolysis of the acetate groups with NH3 in CH3OH resulted in the formation of epoxy alcohol isomers respectively. These epoxy alcohol isomers were subjected to trans-dihydroxylation reaction with acid (H2SO4) in the presence of water to afford triols. Acetylation of the free hydroxyl groups produced benzofuran triacetates in high yields. Ring-opening reaction of furan triacetates with sulfamic acid catalyzed in the presence of acetic acid/acetic anhydrate and subsequently hydrolysis of the acetate groups with ammonia gave the targeted cyclohexane carbasugar-based pentols. All products were separated and purified by chromatographic and crystallographic methods. Structural analyses of all compounds were conducted by spectral techniques including NMR and X-ray analyses. The biological inhibition activity of the target compounds was tested against glycosidase enzymes, α- and ß-glucosidase.

Antileishmanial Carbasugars from Geosmithia langdonii.

Two new carbasugar-type metabolites, (1 S,2 R,3 R,4 R,5 R)-2,3,4-trihydroxy-5-methylcyclohexyl-2',5'-dihydroxybenzoate (1) and (1 S,2 S,3 S,4 R,5 R)-4-[(2',5'-dihydroxybenzyl)oxy]-5-methylcyclohexane-1,2,3-triol (2), were isolated from the filamentous fungus Geosmithia langdonii isolated from cotton textiles from Assiut, Egypt. The structures of 1 and 2 were elucidated based on comprehensive 1D and 2D NMR and MS data. Compounds 1 and 2 showed antileishmanial activity against Leishmania donovani with IC50 values of 100 and 57 µM, respectively. The (1 S,2 R,3 R,4 R,5 R) absolute configuration of carbasugar 1 was assigned via 2D NMR and experimental and calculated electronic circular dichroism (ECD) data. Similarly, the tentative structure of compound 2 was shown to possess a (1 S,2 S,3 S,4 R,5 R) absolute configuration via comparing its experimental ECD data and the specific rotation with 1 as well as examining the energy-minimized 3D computational models of compounds 1 and 2.

Fluoro-Carba-Sugars are Glycomimetic Activators of the glmS Ribozyme.

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and ß-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.

On the Plausibility of Pseudosugar Formation in Cometary Ices and Oxygen-rich Tholins.

We revisit herein the formation and structure of dihydroxy dioxanes, which can be obtained from prebiotically available precursors and can be regarded as primeval sugar surrogates. Previous studies dealing with the heterogeneous composition of interstellar bodies point to the existence of significant amounts of small polyalcohols along with oxygen-containing oligomers. Even though such derivatives did not give rise to nucleosides and oligonucleotides, nor they were incorporated into subsequent metabolic routes, molecular chimeras based on sugar-like species could be opportunistic scaffolds in pre-evolutionary scenarios. We could figure out that pseudosugars, assembled by hemiacetalic bonds from available precursors in both interstellar and terrestrial scenarios, were presumably more abundant than thought. Moreover, these species share some key features with naturally-occurring sugar rings, such as anomeric preferences, coordinating ability, and the prevalent occurrence of racemic compounds.

Synthesis and biological evaluation of phosphoramidate prodrugs of two analogues of 2-deoxy-d-ribose-1-phosphate directed to the discovery of two carbasugars as new potential anti-HIV leads.

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported.

Bicyclic hybrid sugars as glycosidase inhibitors: synthesis and comparative study of inhibitory activities of fused oxa-oxa, oxa-aza, and oxa-carbasugar hybrid molecules.

A few bicyclic hybrid sugar molecules comprising of oxa-aza, oxa-oxa, and oxa-carbasugar fused skeletons were designed and synthesized from C-2 acetoxyglucal involving Ferrier rearrangement, Grignard addition, and ring-closing metathesis as key steps. The inhibitory activities of the synthesized molecules were tested against commercially available enzymes, which revealed the sugar-piperidine and sugar-pyran hybrids as potent and selective inhibitors.

Total syntheses and structural validation of lincitol A, lincitol B, uvacalol I, uvacalol J, and uvacalol K.

Natural carbasugars are an important class of biologically active compounds. Due to their conformational freedom and the subtle difference in spectral characteristics between isomers, often their NMR-based structural assignments are erroneous. It is thus important to validate their structural identity through chemical synthesis. We report the first total syntheses and structural validation of five natural carbasugars, namely, lincitol A, lincitol B, uvacalol I, uvacalol J, and uvacalol K in their racemic forms, from a myo-inositol-derived common intermediate. This intermediate was synthesized by the vinylogous ring opening of myo-inositol orthoester cage under mild acidic conditions in six steps from myo-inositol. From this intermediate, we achieved the syntheses of (±)-lincitol A in six steps, (±)-lincitol B in seven steps, (±)-uvacalol I in five steps, (±)-uvacalol J in five steps, and (±)-uvacalol K in seven steps. The structure and relative stereochemistry of these natural products were confirmed by comparing the (1)H and (13)C NMR spectra of synthesised natural products with the reported data. These syntheses involved several unprecedented protecting-group manipulations and unexpected reactivities.

Fluoro-C-glycosides and fluoro-carbasugars, hydrolytically stable and synthetically challenging glycomimetics.

Fluoro-C-glycosides and fluoro-carbasugars are a particular subclass of hydrolytically stable glycomimetics that are expected to have different, hopefully improved properties thanks to the stereoelectronic features of the fluoroalkyl moiety. This review summarizes the studies devoted to the synthesis of such structures as well as the studies regarding their conformational behaviour and their potential as carbohydrate analogues.

Recent developments in design and synthesis of bicyclic azasugars, carbasugars and related molecules as glycosidase inhibitors.

The importance of glycosidase inhibitors and especially the bicyclic molecules has led to design and assessment of many analogs of naturally occurring molecules. This review focuses on the synthesis and enzyme inhibitions of a few selected (synthetic or non-naturally occurring) molecules that have been reported in the last decade, which allow one to draw some connection between varying the structural features and their effect on glycosidase inhibitions. It is expected that further improvements based on these features could lead to improved inhibitors.

Synthesis of 4-membered carbasugars by way of stereoselective SmI2-mediated aldehyde-alkene cyclization.

A stereodivergent synthesis of the first examples of 4-membered carbasugars has been achieved from vitamin C by way of an efficient intramolecular SmI2-mediated aldehyde-alkene coupling. In this key step, cylobutanes with four contiguous asymmetric centers are generated with a high level of stereocontrol.