Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions.

Carbohydrates (glycans, saccharides, and sugars) are essential molecules in all domains of life. Research on glycoscience spans from chemistry to biomedicine, including material science and biotechnology. Access to pure and well-defined complex glycans using synthetic methods depends on the success of the employed glycosylation reaction. In most cases, the mechanism of the glycosylation reaction is believed to involve the oxocarbenium ion. Understanding the structure, conformation, reactivity, and interactions of this glycosyl cation is essential to predict the outcome of the reaction. In this Account, building on our contributions on this topic, we discuss the theoretical and experimental approaches that have been employed to decipher the key features of glycosyl cations, from their structures to their interactions and reactivity.We also highlight that, from a chemical perspective, the glycosylation reaction can be described as a continuum, from unimolecular SN1 with naked oxocarbenium cations as intermediates to bimolecular SN2-type mechanisms, which involve the key role of counterions and donors. All these factors should be considered and are discussed herein. The importance of dissociative mechanisms (involving contact ion pairs, solvent-separated ion pairs, solvent-equilibrated ion pairs) with bimolecular features in most reactions is also highlighted.The role of theoretical calculations to predict the conformation, dynamics, and reactivity of the oxocarbenium ion is also discussed, highlighting the advances in this field that now allow access to the conformational preferences of a variety of oxocarbenium ions and their reactivities under SN1-like conditions.Specifically, the ground-breaking use of superacids to generate these cations is emphasized, since it has permitted characterization of the structure and conformation of a variety of glycosyl oxocarbenium ions in superacid solution by NMR spectroscopy.We also pay special attention to the reactivity of these glycosyl ions, which depends on the conditions, including the counterions, the possible intra- or intermolecular participation of functional groups that may stabilize the cation and the chemical nature of the acceptor, either weak or strong nucleophile. We discuss recent investigations from different experimental perspectives, which identified the involved ionic intermediates, estimating their lifetimes and reactivities and studying their interactions with other molecules. In this context, we also emphasize the relationship between the chemical methods that can be employed to modulate the sensitivity of glycosyl cations and the way in which glycosyl modifying enzymes (glycosyl hydrolases and transferases) build and cleave glycosidic linkages in nature. This comparison provides inspiration on the use of molecules that regulate the stability and reactivity of glycosyl cations.

Stereoselective Synthesis of 1-C-Diethylphosphonomethyl and -difluoromethyl Iminosugars from Sugar Lactams.

A strategy allowing the straightforward synthesis of 1-C-phosphonomethyl and 1-C-phosphonodifluoromethyl iminosugars is reported. Conversion of sugar lactams to the corresponding imines with Schwartz's reagent followed by their reaction with LiCH2P(O)(OEt)2 and LiCF2P(O)(OEt)2 stereoselectively afforded the 1,2-cis and 1,2-trans glycosyl phosphonates, respectively, in modest to good yields. Application of this methodology to C-2 orthogonally protected sugar lactams paved the way to 2-acetamido- and 2-deoxy-1-C-phosphonomethyl iminosugars.

Iminosugar C-Glycosides Work as Pharmacological Chaperones of NAGLU, a Glycosidase Involved in MPS IIIB Rare Disease*.

Mucopolysaccharidosis type IIIB is a devastating neurological disease caused by a lack of the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU), leading to a toxic accumulation of heparan sulfate. Herein we explored a pharmacological chaperone approach to enhance the residual activity of NAGLU in patient fibroblasts. Capitalizing on the three-dimensional structures of two modest homoiminosugar-based NAGLU inhibitors in complex with bacterial homolog of NAGLU, CpGH89, we have synthesized a library of 17 iminosugar C-glycosides mimicking N-acetyl-D-glucosamine and bearing various pseudo-anomeric substituents of both α- and ß-configuration. Elaboration of the aglycon moiety results in low micromolar selective inhibitors of human recombinant NAGLU, but surprisingly it is the non-functionalized and wrongly configured ß-homoiminosugar that was proved to act as the most promising pharmacological chaperone, promoting a 2.4 fold activity enhancement of mutant NAGLU at its optimal concentration.

Insight into the Ferrier Rearrangement by Combining Flash Chemistry and Superacids.

The transformation of glycals into 2,3-unsaturated glycosyl derivatives, reported by Ferrier in 1962, is supposed to involve an α,ß unsaturated glycosyl cation, an elusive ionic species that has still to be observed experimentally. Herein, while combination of TfOH and flow conditions failed to observe this ionic species, its extended lifetime in superacid solutions allowed its characterization by NMR-based structural analysis supported by DFT calculations. This allyloxycarbenium ion was further exploited in the Ferrier rearrangement to afford unsaturated nitrogen-containing C-aryl glycosides and C-alkyl glycosides under superacid and flow conditions, respectively.

Synthesis and glycosidase inhibition of conformationally locked DNJ and DMJ derivatives exploiting a 2-oxo-C-allyl iminosugar.

A series of analogs of the iminosugars 1-deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (DMJ), in which an extra five or six-membered ring has been fused to the C1-C2 bond have been prepared. The synthetic strategy exploits a key 2-keto-C-allyl iminosugar, easily accessible from gluconolactam, which upon Grignard addition and RCM furnishes a bicyclic scaffold that can be further hydroxylated at the C[double bond, length as m-dash]C bond. This strategy furnished DNJ mimics with the piperidine ring locked in a 1C4 conformation with all substituents in axial orientation when fused to a six-membered ring. Addition of an extra ring to DNJ and DMJ motif proved to strongly modify the glycosidase inhibition profile of the parent iminosugars leading to modest inhibitors. The 2-keto-C-allyl iminosugar scaffold was further used to access N-acetylglycosamine analogs via oxime formation.

Structural and Computational Analysis of 2-Halogeno-Glycosyl Cations in the Presence of a Superacid: An Expansive Platform.

An expansive NMR-based structural analysis of elusive glycosyl cations derived from natural and non-natural monosaccharides in superacids is disclosed. For the first time, it has been possible to explore the consequence of deoxygenation and halogen substitution at the C2 position in a series of 2-halogenoglucosyl, galactosyl, and mannosyl donors in the condensed phase. These cationic intermediates were characterized using low-temperature in situ NMR experiments supported by DFT calculations. The 2-bromo derivatives display intramolecular stabilization of the glycosyl cations. Introducing a strongly electron-withdrawing fluorine atom at C2 exerts considerable influence on the oxocarbenium ion reactivity. In a superacid, these oxocarbenium ions are quenched by weakly coordinating SbF6- anions, thereby demonstrating their highly electrophilic character and their propensity to interact with poor nucleophiles.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

In silico analyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay-Sachs disease.

The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.

4'-Methoxyphenacyl-Assisted Synthesis of ß-Kdo Glycosides.

3-Deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) glycosides are mainly found in capsular polysaccharides and extracellular exopolysaccharides from Gram-negative bacteria. These compounds have profound biological implications in immune response and act as virulence factors. We have developed a novel methodology for the stereoselective synthesis of ß-Kdo glycosides via the use of a 4'-methoxyphenacyl (Phen) auxiliary group at the C1 position of a peracetylated ß-Kdo thioglycoside. Under the promotion of NIS/AgOTf in acetonitrile, a series of Kdo glycosides was synthesized in good yield and ß-selectivity while minimizing the formation of undesirable glycals. Stereoselectivity of the glycosylation was shown to be modulated by various factors such as promotor, solvent, anomeric ratio of donor, nature of acceptor, and Phen substitution. Chemoselective cleavage of the Phen group was performed under the action of Zn/HOAc. DFT calculations together with experimental results suggested that α-triflate and a six-membered α-spiroPhen are plausible intermediates of the reaction, accounting for the enhanced formation of ß-Kdo glycosides. The developed methodology could be applied to the synthesis of ß-Kdo-containing glycans from pathogenic bacteria.

Conformational Plasticity in Glycomimetics: Fluorocarbamethyl-L-idopyranosides Mimic the Intrinsic Dynamic Behaviour of Natural Idose Rings.

Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L-iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free-energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine-containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose- and glucose-like rings. We have used low-temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose-like rings. Under these conditions, the exchange rate becomes slow in the (19) F NMR spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural differences between these compounds, a remarkable difference in their dynamic behaviour indeed occurs. The importance of introducing fluorine atoms in these sugars mimics is also highlighted. Only the use of (19) F NMR spectroscopic experiments has permitted the unveiling of key features of the conformational equilibrium that would have otherwise remained unobserved.

Synthesis of the Tetrasaccharide Repeating Unit of the ß-Kdo-Containing Exopolysaccharide from Burkholderia pseudomallei and B. cepacia Complex.

The synthesis of the repeating unit of the immunogenic ß-Kdo-containing exopolysaccharide produced by Burkholderia pseudomallei and bacteria of the B. cepacia complex is described. The target tetrasaccharide was synthesized via stereoselective 1,2-cis- and 1,2-trans-galactosylations and ß-Kdosylation. A [3 + 1] coupling reaction between a trigalactosyl N-phenyl-2,2,2-trifluoroacetimidate donor and a Kdo acceptor has been successfully achieved for the assembly of the tetrasaccharide skeleton.

Non-specific accumulation of glycosphingolipids in GNE myopathy.

BACKGROUND: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood. METHODS: Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients' fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne(M712T/M712T) knock-in mice. RESULTS: Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations. CONCLUSION: GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.

Intramolecular aglycon delivery enables the synthesis of 6-deoxy-ß-D-manno-heptosides as fragments of Burkholderia pseudomallei and Burkholderia mallei capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia mallei are potential bioterrorism agents. They express the same capsular polysaccharide (CPS), a homopolymer featuring an unusual [→3)-2-O-acetyl-6-deoxy-ß-D-manno-heptopyranosyl-(1→] as the repeating unit. This CPS is known to be one of the main targets of the adaptive immune response in humans and therefore represents a crucial subunit candidate for vaccine development. Herein, the stereoselective synthesis of mono- and disaccharidic fragments of the B. pseudomallei and B. mallei CPS repeating unit is reported. The synthesis of 6-deoxy-ß-D-manno-heptosides was investigated using both inter- and intramolecular glycosylation strategies from thio-manno-heptose that was modified with 2-naphthylmethyl (NAP) at C2. We show here that NAP-mediated intramolecular aglycon delivery (IAD) represents a suitable approach for the stereocontrolled synthesis of 6-deoxy-ß-D-manno-heptosides without the need for rigid 4,6-O-cyclic protection of the sugar skeleton. The IAD strategy is highly modular, as it can be applied to structurally diverse acceptors with complete control of stereoselectivity. Problematic hydrogenation of the acetylated disaccharides was overcome by using a microfluidic continuous flow reactor.

gem-Difluorocarbadisaccharides: restoring the exo-anomeric effect.

Molecular mimicry is an essential part of the development of drugs and molecular probes. In the chemical glycobiology field, although many glycomimetics have been developed in the past years, it has been considered that many failures in their use are related to the lack of the anomeric effects in these analogues. Additionally, the origin of the anomeric effects is still the subject of virulent scientific debates. Herein, by combining chemical synthesis, NMR methods, and theoretical calculations, we show that it is possible to restore the anomeric effect for an acetal when replacing one of the oxygen atoms by a CF2 group. This result provides key findings in chemical sciences. On the one hand, it strongly suggests the key relevance of the stereoelectronic component of the anomeric effect. On the other hand, the CF2 analogue adopts the natural glycoside conformation, which might provide new avenues for sugar-based drug design.

Conformational selection in glycomimetics: human galectin-1 only recognizes syn-Ψ-type conformations of ß-1,3-linked lactose and its C-glycosyl derivative.

The human lectin galectin-1 (hGal-1) translates sugar signals, that is, ß-galactosides, into effects on the level of cells, for example, growth regulation, and has become a model for studying binding of biopharmaceutically relevant derivatives. Bound-state conformations of Galß-C-(1→3)-Glcß-OMe (1) and its ßGal-(1→3)-ßGlc-OMe disaccharide parent compound were studied by using NMR spectroscopy (transferred (TR)-NOESY data), assisted by docking experiments and molecular dynamics (MD) simulations. The molecular recognition process involves a conformational selection event. Although free C-glycoside access four distinct conformers in solution, hGal-1 recognizes shape of a local minimum of compound 1, the syn-Φ/syn-Ψ conformer, not the structure at global minimum. MD simulations were run to explain, in structural terms, the observed geometry of the complex.

C-branched iminosugars: α-glucosidase inhibition by enantiomers of isoDMDP, isoDGDP, and isoDAB-L-isoDMDP compared to miglitol and miglustat.

The Ho crossed aldol condensation provides access to a series of carbon branched iminosugars as exemplified by the synthesis of enantiomeric pairs of isoDMDP, isoDGDP, and isoDAB, allowing comparison of their biological activities with three linear isomeric natural products DMDP, DGDP, and DAB and their enantiomers. L-IsoDMDP [(2S,3S,4R)-2,4-bis(hydroxymethyl)pyrrolidine-3,4-diol], prepared in 11 steps in an overall yield of 45% from d-lyxonolactone, is a potent specific competitive inhibitor of gut disaccharidases [K(i) 0.081 µM for rat intestinal maltase] and is more effective in the suppression of hyperglycaemia in a maltose loading test than miglitol, a drug presently used in the treatment of late onset diabetes. The partial rescue of the defective F508del-CFTR function in CF-KM4 cells by L-isoDMDP is compared with miglustat and isoLAB in an approach to the treatment of cystic fibrosis.

Skeletal rearrangement of seven-membered iminosugars: synthesis of (-)-adenophorine, (-)-1-epi-adenophorine and derivatives and evaluation as glycosidase inhibitors.

The mirror image of natural product (+)-adenophorine along with its 1-epi-, 1-homo-analogs and other derivatives have been synthesized and evaluated as glycosidase inhibitors. The synthetic strategy is based on the skeletal rearrangement of tetrahydroxylated C-alkyl azepanes obtained via a Staudinger/azaWittig/alkylation sequence starting from a sugar-derived azidolactol. Several organometallic species have been investigated for the alkylation step including organomagnesium, organolithium, organozinc, organoaluminum and organocerium reagents. While diallylzinc proved to be the most efficient to introduce an allyl substituent, disappointing results were obtained with the other organometallic species leading either to lower yields or no reaction. Enzymatic assays indicate that (-)-adenophorine is a moderate α-l-fucosidase inhibitor.

An "against the rules" double bank shot with diisobutylaluminum hydride to allow triple functionalization of α-cyclodextrin.

Frustration leads to overreaction: when diametrically opposed regioselective debenzylation is frustrated, an unexpected double debenzylation reaction affords original tetrafunctionalized cyclodextrins in a controlled and efficient manner. A rationale of the reaction is proposed based on a kinetic study.

Evaluation of nonnatural L-iminosugar C,C-glycosides, a new class of C-branched iminosugars, as glycosidase inhibitors.

Capitalizing on a previously developed Staudinger/azaWittig/Grignard (SAWG)-ring contraction sequence that furnished protected six-membered L-iminosugar C,C-glycosides bearing an allyl group and various substituents at the pseudoanomeric position, the synthesis and glycosidase inhibition of a small library of six- and seven-membered L-iminosugar C,C-glycosides is reported. Their hydrogenolysis or cyclization by RCM followed by deprotection afforded eleven L-iminosugars including spirocyclic derivatives. All compounds adopt a 1C4 conformation in solution according to NMR data. Compared to previously reported branched L-iminosugars, the L-iminosugar C,C-glycosides reported herein were less potent glycosidase inhibitors. However, some of these compounds showed micromolar inhibition of human lysosome ß-glucocerebrosidase suggesting that such iminosugars could be useful to access potent CGase inhibitors by adjusting the structure/length of the pseudoanomeric substituents.

Towards a stable noeuromycin analog with a D-manno configuration: synthesis and glycosidase inhibition of D-manno-like tri- and tetrahydroxylated azepanes.

Noeuromycin is a highly potent albeit unstable glycosidase inhibitor due to its hemiaminal function. While stable D-gluco-like analogs have been reported, no data are available for D-manno-like structures. A series of tri- and tetrahydroxylated seven-membered iminosugars displaying either a D-manno-or a L-gulo-like configuration, were synthesized from methyl α-D-mannopyranoside using a reductive amination-mediated ring expansion as the key step. Screening towards a range of commercial glycosidases demonstrated their potency as competitive glycosidase inhibitors while cellular assay showed selective albeit weak glycoprotein processing mannosidase inactivation.