Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Chemical Synthesis of a Densely Functionalized Aminodisaccharide from Fusobacterium nucleatum ATCC 23726 O-Antigen.

Fusobacterium nucleatum, a colorectal-cancer-associated oncomicrobe, can trigger or accelerate numerous pathologies. We report the first synthesis of a conjugation-ready disaccharide containing six amino groups from F. nucleatum ATCC 23726 O-antigen. Rare 2,3-diamido-d-glucuronic acid amide and 2-acetamido-4-amino-d-fucose were synthesized from d-glucosamine through configuration inversion, nucleophilic substitution, C6 oxidation, and C6 deoxygenation. A judicious choice of protecting groups and reaction conditions enabled the selective installation of N-acetyl, N-propanoyl, N-formyl, and carboxamido groups.

Activation of glycosyl methylpropiolates by TfOH.

Activation of glycosyl methylpropiolates by TfOH was investigated. Armed and superarmed glycosyl donors can be activated by use of 0.2 equivalent TfOH whereas 1.0 equivalent of TfOH was required for the activation of the disarmed glycosyl donors. All the glycosidations gave very good yields. The method is suitable for synthesis of glycosides and disaccharides and it may result in the hydrolysis of the interglycosidic bond if the sugar at the non-reducing end is armed or superarmed. These problems are not seen when gold-catalyzed activation procedures are invoked for the activation of glycosyl alkynoates.

Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

Site-selective, stereocontrolled glycosylation of minimally protected sugars.

The identification of general and efficient methods for the construction of oligosaccharides stands as one of the great challenges for the field of synthetic chemistry1,2. Selective glycosylation of unprotected sugars and other polyhydroxylated nucleophiles is a particularly significant goal, requiring not only control over the stereochemistry of the forming bond but also differentiation between similarly reactive nucleophilic sites in stereochemically complex contexts3,4. Chemists have generally relied on multi-step protecting-group strategies to achieve site control in glycosylations, but practical inefficiencies arise directly from the application of such approaches5-7. Here we describe a strategy for small-molecule-catalyst-controlled, highly stereo- and site-selective glycosylations of unprotected or minimally protected mono- and disaccharides using precisely designed bis-thiourea small-molecule catalysts. Stereo- and site-selective galactosylations and mannosylations of a wide assortment of polyfunctional nucleophiles is thereby achieved. Kinetic and computational studies provide evidence that site-selectivity arises from stabilizing C-H/π interactions between the catalyst and the nucleophile, analogous to those documented in sugar-binding proteins. This work demonstrates that highly selective glycosylation reactions can be achieved through control of stabilizing non-covalent interactions, a potentially general strategy for selective functionalization of carbohydrates.

A gulose moiety contributes to the belomycin (BLM) disaccharide selective targeting to lung cancer cells.

Eight mono- or disaccharide analogues derived from BLM disaccharide, along with the corresponding carbohydate-dye conjugates have been designed and synthesized in this study, aiming at exploring the effect of a gulose residue on the cellular binding/uptake of BLM disaccharide and it possible uptake mechanism. Our evidence is presented indicating that, for the cellular binding/uptake of BLM disaccharide, a gulose residue is an essential subunit but unrelated to its chemical nature. Interestingly, d-gulose-dye conjugate is able to selectively target A549 cancer cells, but l-gulose-dye conjugate fails. Further uptake mechanism studies demonstrate d-gulose-dye derivatives similar to BLM disaccharide-dye ones behave in a temperature- and ATP-dependent manner, and are partly directed by the GLUT1 receptor. Moreover, d-gulose modifying gemcitabine 53a exhibits more potent antitumor activity compared to derivatives 53b-c in which gemcitabine is decorated with other monosaccharides. Taken together, the monosacharide d-gulose conjugate offers a new strategy for solving cytotoxic drugs via the increased tumor targeting in the therapy of lung cancer.

Light-Mediated Cross-Coupling of Anomeric Trifluoroborates.

Stereoselective reactions at the anomeric carbon constitute the cornerstone of preparative carbohydrate chemistry. Here, we report stereoselective C-arylation and etherification reactions of anomeric trifluoroborates derived from BMIDA esters. These reactions are characterized by high anomeric selectivities for 2-deoxysugars and broad substrate scope (24 examples), including disaccharides and trifluoroborates with free hydroxyl groups. Taken together, this new class of carbohydrate reagents adds the palette of anomeric nucleophile reagents suitable for efficient installation of C-C bonds.

Synthesis, Structure-Activity Relationships, and In Vivo Evaluation of Novel Tetrahydropyran-Based Thiodisaccharide Mimics as Galectin-3 Inhibitors.

Galectin-3 is a member of a family of ß-galactoside-binding proteins. A substantial body of literature reports that galectin-3 plays important roles in cancer, inflammation, and fibrosis. Small-molecule galectin-3 inhibitors, which are generally lactose or galactose-based derivatives, have the potential to be valuable disease-modifying agents. In our efforts to identify novel galectin-3 disaccharide mimics to improve drug-like properties, we found that one of the monosaccharide subunits can be replaced with a suitably functionalized tetrahydropyran ring. Optimization of the structure-activity relationships around the tetrahydropyran-based scaffold led to the discovery of potent galectin-3 inhibitors. Compounds 36, 40, and 45 were selected for further in vivo evaluation. The synthesis, structure-activity relationships, and in vivo evaluation of novel tetrahydropyran-based galectin-3 inhibitors are described.

Site-Selective Acylations of α- and ß-Hydroxyamides in Complex Molecules: Application of Template-Driven Acylation to Disaccharides and a Glycopeptide.

Site-selective acylations of α-and ß-hydroxyamides in complex polyols are described. The combination of a pyridine aldoxime ester and Zn(OTf)2 facilitates the acylation of two types of N-glycolyl disaccharides, namely, Gal-GlcNGc and Neu5Gc-Gal, both of which are partial structures of polysaccharides responsible for biological actions, with highly site-selective modifications achieved. Furthermore, biotinylation, one of the most important techniques in chemical biology, is used to site-selectively acylate the ß-hydroxyl group in a glycopeptide.

Palladium(II)-assisted activation of thioglycosides.

Described herein is the first example of glycosidation of thioglycosides in the presence of palladium(ii) bromide. While the activation with PdBr2 alone was proven feasible, higher yields and cleaner reactions were achieved when these glycosylations were performed in the presence of propargyl bromide as an additive. Preliminary mechanistic studies suggest that propargyl bromide assists the reaction by creating an ionizing complex, which accelerates the leaving group departure. A variety of thioglycoside donors in reactions with different glycosyl acceptors were investigated to determine the initial scope of this new reaction. Selective and chemoselective activation of thioglycosides over other leaving groups has also been explored.

New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis and SPR-based binding inhibition assays.

The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has been implicated in the early stages of many infection processes. DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent potentially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antagonists, mimics of the natural moiety, characterized by the presence of a real d-carbamannose unit which confers greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important information about the influence of the lipophilic substituents present in specific positions of the carba scaffold. Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a more favorable IC50 value than those of the previously described real carba-analogues. This study provides valuable knowledge for the implementation of further structural modifications towards improved inhibitors.

Characterization of crystalline forms of gaxilose, a diagnostic drug.

Lactose intolerance is a pathology caused by lactase enzyme deficiency, usually produced in the intestinal cells provoking symptoms as abdominal pain, bloating, diarrhea, gas and nausea. Gaxilose, 4-O-ß-D galactopyranosyl-d-xylose, is used as a diagnostic drug for a non-invasive method for hypolactasia diagnosis. To date, no definitive guide for identifying gaxilose and distinguishing between crystalline forms is available. Data have been collected from a number of different analytical techniques in order to provide a full characterization of the compound and a simple method to discriminate between two solid forms.

Synthetic Studies on the Incorporation of N-Acetylallosamine in Hyaluronic Acid-Inspired Thiodisaccharides.

Two approaches for the synthesis of the thiodisaccharide ß-S-GlcA(1→3)ß-S-AllNAc are described here. The target disaccharide was a C-3 epimer and thio-analogue of the hyaluronic acid repetitive unit, tuned with a thiopropargyl anomeric group for further click conjugation. Thus, we analysed and tested two convenient sequences, combining the two key steps required to introduce the thioglycosidic bonds and consequently reach the target molecule: the SN2 substitution of a good leaving group (triflate) present at C-3 of a GlcNAc derivative and the introduction of the anomeric thiopropargyl substituent. The use of a 2-azido precursor showed to be a convenient substrate for the SN2 step. Nevertheless, further protecting group manipulation and the introduction of the thiopropargyl anomeric residue were then required. This approach showed to provide access to a variety of thiodisaccharide derivatives as interesting building blocks for the construction of neoglycoconjugates.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

Novel disaccharide inhibitors for the bacterial galactosyltransferase LgtC: Design, synthesis via Heyns rearrangement, and biochemical evaluation.

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors.

S-Glycosides: synthesis of S-linked arabinoxylan oligosaccharides.

S-Glycosides are important tools for the elucidation of specific protein-carbohydrate interactions and can significantly aid structural and functional studies of carbohydrate-active enzymes, as they are often inert or act as enzyme inhibitors. In this context, this work focuses on the introduction of an S-linkage into arabinoxylan oligosaccharides (AXs) in order to obtain a small collection of synthetic tools for the study of AXs degrading enzymes. The key step for the introduction of the S-glycosidic linkage involved anomeric thiol S-alkylation of an orthogonally protected l-arabinopyranoside triflate. The resulting S-linked disaccharide was subsequently employed in a series of glycosylation reactions to obtain a selectively protected tetrasaccharide. This could be further elaborated through chemoselective deprotection and glycosylation reactions to introduce branching l-arabinofuranosides.

Metal-free synthesis of imino-disaccharides and calix-iminosugars by photoinduced radical thiol-ene coupling (TEC).

Radical thiol-ene coupling was exploited for the first time to prepare imino-disaccharides and multivalent iminosugars starting from sugar thiols and iminosugar alkenes or iminosugar thiols and tetra-allylated calixarene, respectively.

High-Affinity N-(2-Hydroxypropyl)methacrylamide Copolymers with Tailored N-Acetyllactosamine Presentation Discriminate between Galectins.

N-Acetyllactosamine (LacNAc; Galß4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by ß-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.

Synthesis of 3-O-Sulfated Disaccharide and Tetrasaccharide Standards for Compositional Analysis of Heparan Sulfate.

3-O-Sulfation on the glucosamine sugar unit in heparan sulfate (HS) is linked to various biological functions, including the anticoagulant activity to treat thrombotic disorders in hospitals. The 3-O-sulfated glucosamine is biosynthesized by heparan sulfate glucosamine 3-sulfotransferases. Because of its biological significance, there is a need for 3-O-sulfated oligosaccharide standards to facilitate the compositional analysis of HS. These oligosaccharides must contain a Δ4,5-unsaturated uronic acid (ΔUA) residue at the nonreducing end, which is due to the depolymerization reaction catalyzed by heparin lyases used during the compositional analysis procedure. Here, we describe a protocol for the preparation of one 3-O-sulfated disaccharide (compound 4) and three 3-O-sulfated tetrasaccharides (compound 1-3) in a milligram scale. The synthesis of 3-O-sulfated disaccharide and tetrasaccharide standards was completed by degrading synthetic octasaccharides using heparin lyases. Further analysis revealed that 3-O-sulfated oligosaccharide standards are labile under basic conditions, confirming the findings from a previous study. The unwanted degradation was reduced by decreasing the pH in the presence of phosphate buffer. The 3-O-sulfated oligosaccharide standards are reagents to characterize 3-O-sulfation in HS derived from biological sources.

Construction of sulfur-containing moieties in the total synthesis of natural products.

Covering: January 2013 to September 2018Sulfur-containing natural products are a large class of significant functional molecules. Many of these compounds exhibit potent biological activities and pharmacological properties; in fact, some of them have been developed into important drugs. The total synthesis of sulfur-containing natural products is a subject that has long attracted significant attention from synthetic organic chemists; to achieve this goal, various methods have been developed over the past years. This review surveys total syntheses of sulfur-containing natural products that introduce sulfur atoms using different sulfurization agents to construct related sulfur-containing moieties.