Protecting-Group-Free Synthesis of ADP-Ribose and Dinucleoside Di-/Triphosphate Derivatives via P(V)-P(V) Coupling Reaction.

Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through in situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.

Convergent synthesis of functionalized derivatives of stage-specific embryonic antigens 3 & 4.

Stage-specific embryonic antigens (SSEAs) are carbohydrate markers that have diverse roles in embryonic development. However, the exact roles of SSEAs remain unclear. To obtain mechanistic insights into their roles, we aimed to develop functionalized SSEA glycan analogs via chemical synthesis. Herein, we report a convergent synthetic approach for SSEA-3 and SSEA-4 analogs using readily available versatile building blocks. A key step, namely the stereoselective glycosylation of a common tetrasaccharide acceptor, was successfully achieved using a 4-O-Bn Gal donor for SSEA-3 and a Neu-Gal donor for SSEA-4, which were previously developed by our group. The obtained SSEA-3 and SSEA-4 glycans were further functionalized with biotin and deuterated lipid for applications in biological studies. Thus, the findings of this study will facilitate further research on SSEAs.

Unprecedented neighboring group participation of C2 N-imidoxy functionalities for 1,2-trans-selective glycosylation.

Stereoselective glycosylation reactions are important in carbohydrate chemistry. The most used method for 1,2-trans(ß)-selective glycosylation involves the neighboring group participation (NGP) of the 2-O-acyl protecting group; nevertheless, an alternative stereoselective method independent of classical NGP would contribute to carbohydrate chemistry, despite being challenging to achieve. Herein, a ß-selective glycosylation reaction employing unprecedented NGP of the C2 N-succinimidoxy and phthalimidoxy functionalities is reported. The C2 functionalities provided the glycosylated products in high yields with ß-selectivity. The participation of the functionalities from the α face of the glycosyl oxocarbenium ions gives stable six-membered intermediates and is supported by density functional theory calculations. The applicability of the phthalimidoxy functionality for hydroxyl protection is also demonstrated. This work expands the scope of functionalities tolerated in carbohydrate chemistry to include O-N moieties.

Fluorescent GD2 analog for single-molecule imaging.

Ganglioside GD2 is associated with the proliferation and migration of breast cancer cells. However, the precise role of GD2 is unclear because its tendency to form dynamic and transient domains in cell plasma membranes (PMs), called lipid rafts, makes it difficult to observe. Previously, we developed fluorescent analogs of gangliosides (e.g., GM3 and GM1), which enabled the observation of lipid raft formation for the first time using single-molecule imaging. In this report, we describe the first chemical synthesis of a fluorescent ganglioside, GD2. A biophysical analysis of the synthesized analog revealed its raft-philic character, suggesting its potential to aid single-molecule imaging-based investigations into raft-associated interactions.

Chemical synthesis of sialoglyco-architectures.

The synthesis of sialic acid-containing molecules has posed a formidable challenge to carbohydrate chemists for over 50 years. Our research group has intensively searched for robust chemistry to enable the construction of a broad spectrum of sialic acid-containing molecules to advance the understanding and application of their biological functions. Herein, we describe our research findings on the development of sialic acid donors for α-selective glycosidation and the chemical synthesis of sialic acid- containing molecules, with a special focus on gangliosides and their fluorescent probes.

Full Stereocontrol in α-Glycosidation of 3-Deoxy-d-manno-2-octulosonic Acid (Kdo) Using Macrobicyclic Glycosyl Donors.

We describe a method for the α-selective glycosidation of 3-deoxy-d-manno-2-octulosonic acid (Kdo) using a macrobicyclic Kdo donor as the precursor of a bridgehead oxocarbenium ion, whose stereoselectivity is not affected by the substrate structure and reaction conditions. Strapping Kdo via tethering in the α-configuration at the C1 and C5 positions completely blocked nucleophilic attack to the ß-face of the anomeric center by sterically hindering the bicyclic system, realizing full α-selectivity during glycosidation.

Straightforward Synthesis of the Poly(ADP-ribose) Branched Core Structure.

Poly(ADP-ribosyl)ation is a post-translational modification that produces poly(ADP-ribose) with a branched structure every 20-50 units; such branching structure has been previously suggested to be involved in regulating chromatin remodeling. To elucidate its detailed functions, we developed a straightforward method for the synthesis of the poly(ADP-ribose) branched core structure, α-d-ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2')-adenosine 5',5'',5‴-trisphosphate 1, from 6-chloropurine ribofuranoside 4 in 10 steps and 6.1% overall yield. The structure poses synthetic challenges for constructing iterative α-1,2-cis-glycosidic bonds in the presence of a purine base and the installation of three phosphate groups at primary hydroxyl groups. Iterative glycosidic bonds were formed by α-1,2-cis-selective ribofuranosylation using 2-O-(2-naphthylmethyl)-protected thioglycoside donor 6 and a thiophilic bismuth promoter. After the construction of diribofuranosyl adenosine 5 had been constructed, it was chemo- and regioselectively phosphorylated at a later stage. Subsequent deprotection provided the synthetic target 1.

Development of lacto-series ganglioside fluorescent probe using late-stage sialylation and behavior analysis with single-molecule imaging.

Gangliosides are a family of sialic-acid-containing glycosphingolipids that form dynamic domains (lipid rafts) with proteins in cell plasma membranes (PMs), and are involved in various biological processes. The dynamic behavior of gangliosides can be elucidated by analyzing fluorescently-labeled molecules with a powerful technique known as single-molecule imaging. We previously developed fluorescent probes for ganglioside subfamilies such as the ganglio- and globo-series, and investigated their behavior in cell PMs. This study targeted a lacto-series ganglioside, sialyl-lactotetraosylceramide, whose behavior in PMs has not yet been investigated. We applied a recently reported method for the direct sialylation of oligosaccharyl lipid acceptors to synthesize the fluorescent ganglioside probes. The glycolipid acceptor exhibited high solubility in organic solvents owing to the installation of a large quantity of p-tert-butylbenzoyl protecting groups, which ensured direct α-sialylation at relatively low temperatures. Biophysical evaluation of the synthesized probe determined that it behaved as a raft molecule in cell PMs. Furthermore, single-molecule imaging revealed cis interactions between the lacto-series ganglioside and a major raft molecule (GPI-anchored protein CD59). Moreover, the fluorescent non-sialylated (asialyl) lactotetraosylceramide behaved similarly to its sialyl counterpart.

Protecting-group- and microwave-free synthesis of ß-glycosyl esters and aryl ß-glycosides of N-acetyl-d-glucosamine.

A protecting-group-free method for synthesis of ß-glycosyl esters and aryl ß-glycosides was developed by using latent chemical reactivity of N-acetyl-d-glucosamine (GlcNAc) oxazoline. The GlcNAc oxazoline was spontaneously reacted with carboxylic acids and phenol derivatives via the oxazoline ring opening without the use of a catalyst or heating conditions (i.e., microwave irradiation), affording the desired products in moderate to excellent yields with ß-selectivity. This simple protecting-group-free method exhibits a wide substrate scope and good functional group tolerance, and it allows the efficient production of a novel class of GlcNAc-conjugated biomaterials and prodrug candidates.

Structure-based design of UDP-GlcNAc analogs as candidate GnT-V inhibitors.

BACKGROUND: N-Glycan branching regulates various functions of glycoproteins. N-Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N-glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors. METHODS: We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N-glycan-branching GlcNAc transferases in which GnT-I-V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity. RESULTS: Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action. CONCLUSIONS: Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V. GENERAL SIGNIFICANCE: Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate.

Blockwise synthesis of polylactosamine fragments and keratan sulfate oligosaccharides comprised of dimeric Galß(1 → 4)GlcNAc6Sß.

In this paper, the chemical synthesis of polylactosamine fragments up to docosasaccharide (22-mer) via the blockwise synthetic approach is reported. We used suitably protected tetrasaccharide and octasaccharide sequences as key building blocks. The use of such large building blocks as glycosyl donors and acceptors enabled the rapid construction of polysaccharide frameworks. Furthermore, the coupling reaction between these large building blocks facilitated the purification of glycosylated products, for which size exclusion column chromatography is highly effective. Then, we applied the building blocks to the synthesis of keratan sulfate glycan, which is partially sulfated poly-N-acetyllactosamine. Consequently, we achieved the synthesis of the octasaccharide of a keratan sulfate glycan comprised of a repeating Galß(1 â†’ 4)GlcNAc6Sß disaccharide unit.

Investigation of the Protection of the C4 Hydroxyl Group in Macrobicyclic Kdo Donors.

Chemical synthesis of 3-deoxy-d-manno-2-octulosonic acid (Kdo)-containing glycans, such as bacterial lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs), is in high demand for the development of vaccines against pathogenic bacteria. We have recently achieved the complete α-stereoselective glycosidation of Kdo using a macrobicyclic donor tethered at the C1 and C5 positions. In this study, to expand the scope of Kdo glycosidation, we sought to protect the 4-OH group, thereby shortening the reaction time and ensuring the conversion of the glycosyl acceptor via its selective removal. The protection of the 4-OH group influenced the reactivity of the Kdo donor, and the triisopropylsilyl (TIPS) group acted as a selectively removable booster. The 4-O-TIPS donor allowed the synthesis of the α(2,4)-linked dimeric Kdo sequence, which is widely found in bacterial LPSs.

Advanced Chemical Methods for Stereoselective Sialylation and Their Applications in Sialoglycan Syntheses.

Sialic acid is an important component of cell surface glycans, which are responsible for many vital body functions and should therefore be thoroughly studied to understand their biological roles and association with disorders. The difficulty of isolating large quantities of homogenous-state sialoglycans from natural sources has inspired the development of the corresponding chemical synthesis methods affording acceptable purities, yields, and amounts. However, the related syntheses are challenging because of the difficulties in α-glycosylation of sialic acid, which arises from its certain structural features such as the absence of a stereodirecting group at the C3 position and presence of carboxyl group at the anomeric position. Moreover, the structural complexities of sialoglycans with diverse numbers and locations of sialic acid on the glycan chains pose additional barriers. Thus, efficient α-stereoselective routes to sialosides remain highly sought after, although various types of sialyl donors/acceptors have been developed for the straightforward synthesis of α-sialosides. Herein, we review the latest progress in the α-stereoselective synthesis of sialosides and their applications in the preparation of gangliosides and other sialoglycans.

Stereoselective Synthesis of Diglycosyl Diacylglycerols with Glycosyl Donors Bearing a ß-Stereodirecting 2,3-Naphthalenedimethyl Protecting Group.

Diglycosyl diacylglycerols (DGDGs) are major components of Gram-positive bacterial plasma membranes and are involved in the immune response systems. The chemical synthesis of DGDGs has been highly demanded, as it will allow the elucidation of their biological functions at the molecular level. In this study, we have developed a novel ß-stereodirecting 2,3-naphthalenedimethyl (NapDM) protecting group that is orthogonal to protecting groups commonly used in oligosaccharide synthesis. The NapDM group can be easily cleaved under TFA-mediated acidic conditions. Futhermore, we demonstrated the application of this protecting group to an acyl protecting-group-free strategy by utilizing the NapDM group for the synthesis of DGDGs. This strategy features the use of the ß-stereodirecting NapDM group as an acid-cleavable permanent protecting group and late-stage glycosylation of monoglycosyl diacylglycerol acceptors, enabling the stereoselective synthesis of three different bacterial DGDGs with unsaturated fatty acid chain(s).

Development of Fluorescent Ganglioside GD3 and GQ1b Analogs for Elucidation of Raft-Associated Interactions.

b-Series gangliosides are abundant in central nervous tissues and are involved in important nerve processes. However, their functions are complicated because of their properties of forming dynamic domains in cell plasma membranes (PMs), called lipid rafts. In this study, we aim to develop fluorescently labeled b-series gangliosides that are useful for single-molecule imaging. The chemical synthesis of fluorescent GD3 and GQ1b was achieved using sialylation and ganglioside synthetic methods previously developed by our group. Furthermore, biophysical evaluations demonstrated that synthesized fluorescent GD3 and GQ1b behaved as raft molecules on cell PMs, suggesting their applicability to the study of raft-associated interactions.

Indirect synthetic route to α-l-fucosides via highly stereoselective construction of α-l-galactosides followed by C6-deoxygenation.

We developed an indirect synthetic method for α-l-fucosides. Based on the fact that l-fucose is 6-deoxy-l-galactose, our strategy consists of the stereoselective construction of α-l-galactoside and its conversion to α-l-fucoside via C6-deoxygenation. The formation of α-l-galactoside is strongly directed using 4,6-O-di-tert-butylsilylene(DTBS)-protected l-galactosyl donors. The DTBS-directed α-l-galactosylation showed broad substrate applicability along with excellent coupling yield and α-selectivity. In the C6-deoxygenation of α-l-galactosides, the Barton-McCombie reaction facilitated the conversion to l-fucosides with good yield. To demonstrate the applicability of our method, we synthesized naturally occurring α-l-fucosides.

Efficient diversification of GM3 gangliosides via late-stage sialylation and dynamic glycan structural studies with 19F solid-state NMR.

Sialic acid-containing glycoconjugates are involved in important biological processes such as immune response, cancer metastasis, and viral infection. However, their chemical syntheses have been challenging, mainly due to the difficulties in the α-sialylation of oligosaccharides. Very recently, we established a completely stereoselective sialidation method using a macrobicyclic sialyl donor. Herein, we describe a rational and efficient synthesis of sialoglycolipids via direct sialylation of a glycolipid at a late-stage, based on our novel sialidation method. The synthetic method enabled the development of GM3 ganglioside analogs with various C5-modifications of the sialosyl moiety. Furthermore, the synthesized analog was subjected to solid-state 19F NMR analysis on the model membranes and it revealed the influence of cholesterol on glycan dynamics.

Development of Fluorescently Labeled SSEA-3, SSEA-4, and Globo-H Glycosphingolipids for Elucidating Molecular Interactions in the Cell Membrane.

Glycosphingolipids (GSLs), such as the globo-series GSLs stage-specific embryonic antigen 3 (SSEA-3), SSEA-4, and Globo-H, are specifically expressed on pluripotent stem cells and cancer cells, and are known to be associated with various biological processes such as cell recognition, cell adhesion, and signal transduction. However, the behavior and biological roles of these GSLs are still unclear. In our previous study, we observed the interactions between the lipid raft and GSLs in real-time using single-molecule imaging, where we successfully synthesized various fluorescent analogs of GSLs (e.g., GM1 and GM3). Here, we have developed fluorescent analogs of SSEA-3, SSEA-4, and Globo-H using chemical synthesis. The biophysical properties of these analogs as raft markers were examined by partitioning giant plasma membrane vesicles from RBL-2H3 cells into detergent-resistant membrane fractions and liquid-ordered/liquid-disordered phases. The results indicated that the analogs were equivalent to native-type GSLs. The analogs could be used to observe the behavior of globo-series GSLs for detailing the structure and biological roles of lipid rafts and GSL-enriched nanodomains during cell differentiation and cell malignancy.

Synthesis and Glycan-Protein Interaction Studies of Se-Sialosides by 77Se NMR.

To expand the potential of Se-carbohydrates for multifunctional mimicry of sugars, herein we addressed the synthesis of the highly challenging and biologically significant Se-glycosides of sialic acid (Se-sialosides). An α-sialyl selenolate anion generated in situ smoothly reacted with electrophiles to give α-Se-sialosides as single stereoisomers. A Se-sialoside was sequentially incorporated with selenium, producing a triseleno-sialoside. This molecule was used as a 77Se NMR-active handle for studying glycan-protein interaction, revealing different binding profiles of sialic acid binding proteins.

Chemical synthesis of diglucosyl diacylglycerols utilizing glycosyl donors with stereodirecting cyclic silyl protective groups.

Chemical syntheses of the bacterial diglucosyl diacylglycerols 1-heptadecanoyl-2-pentadecanoyl-3-O-[6-O-(ß-d-glucopyranosyl)-ß-d-glucopyranosyl]-sn-glycerol and 1-(cis-13-octadecenoyl)-2-palmitoyl-3-O-[2-O-(α-d-glucopyranosyl)-α-d-glucopyranosyl]-sn-glycerol are described. The syntheses feature the stereoselective construction of glycosidic linkages in glycosylation reaction by utilizing glycosyl donors with stereodirecting cyclic silyl protective groups. The 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS) group was used for formation of the ß-glycosidic linkage, while the di-tert-butylsilylene (DTBS) group was used for α-linkage formation. The silyl protective groups were chemoselectively cleavable without affecting acyl functionalities on the glycerol moiety and proved effective for the synthesis of diacylglycoglycerolipids.