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.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

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.

Disposition of E-selectin-targeting liposomes in tumor spheroids with a perfusable vascular network.

We constructed tumor spheroids with a perfusable vascular network to assess drug delivery systems that target the tumor vasculature. A tricultured tumor spheroid containing human umbilical vein endothelial cells (HUVECs) was placed in the central compartment of a microfluidic device, and the HUVECs were seeded into the microslit channels on both sides. Angiogenic sprouts began to form within a few days, from both the tumor spheroids and microchannels, and became more abundant and branched, while attracting each other, over time. A continuous vascular network of HUVECs was fully formed on Day 7. The uptake of 3'-(1-carboxy)ethyl sialyl Lewis X mimic (3'-CE sLeX mimic) liposomes, which have previously been proven to recognize E-selectin, in vascular-perfusable tumor spheroids was assessed. 3'-CE sLeX mimic and pegylated liposomes were rarely taken up, but when the vascular network was pretreated with TNF-α and IL-1ß, 3'-CE sLeX mimic liposomes accumulated considerably more in endothelial cells and their vicinity. Taken together, along with the known in vivo expression of E-selectin in tumor angiogenic blood vessels, these results suggest that 3'-CE sLeX mimic liposomes are a promising carrier for targeting tumor vasculature. Furthermore, proinflammatory cytokine treatment may be appropriate for use with vascular-perfusable tumor spheroids in pharmacokinetic studies.

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.

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.

Analysis of biochemical features of ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase (St8sia) 5 isoforms.

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.

FRET detects lateral interaction between transmembrane domain of EGF receptor and ganglioside GM3 in lipid bilayers.

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

The Protein Tyrosine Phosphatase SHP-1 (PTPN6) but Not CD45 (PTPRC) Is Essential for the Ligand-Mediated Regulation of CD22 in BCR-Ligated B Cells.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes α2,6 sialic acid as a specific ligand and interacts with α2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligation-induced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22-mediated signal inhibition, upon BCR ligation in CD45-/- as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP-1-mediated dephosphorylation of CD22 in CD22 clusters.

ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.

ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.

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.

Linear synthesis of the Chol-1 ganglioside core tetrasaccharide and disialyl T antigen glycan using a 5-ureido-sialyl donor.

Herein we describe the linear synthesis of a tetrasaccharyl sialoglycan found in both the Chol-1 ganglioside core and disialyl T antigen. The synthesis featured sialylation with a C5-ureido-modified sialyl donor followed by selective isolation of the desired α-sialoside via 1,5-lactamization. This methodology enables the linear synthesis of sialoglycans and provides practical access to biologically important carbohydrate molecules.

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging.

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40ms and 12ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E-selectin-Mediated Targeting to Inflamed Endothelial Cells.

Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM sLeX mimic liposome.

The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the ß-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

Efficient Synthesis of the Lewis A Tandem Repeat.

The convergent synthesis of the Lewis A (Le(a)) tandem repeat is described. The Le(a) tandem repeat is a carbohydrate ligand for a mannose binding protein that shows potent inhibitory activity against carcinoma growth. The Le(a) unit, {ß-d-Gal-(1→3)-[α-l-Fuc-(1→4)]-ß-d-GlcNAc}, was synthesized by stereoselective nitrile-assisted ß-galactosylation with the phenyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-ß-galactoside, and ether-assisted α-fucosylation with fucosyl (N-phenyl)trifluoroacetimidate. This common Le(a) unit was easily converted to an acceptor and donor in high yields, and the stereoselective assembly of the hexasaccharide and dodecasaccharide as the Le(a) tandem repeat framework was achieved by 2-trichloroacetamido-assisted ß-glycosylation and the (N-phenyl)trifluoroacetimidate method.

Structure-Activity Relationship Study of the Neuritogenic Potential of the Glycan of Starfish Ganglioside LLG-3 (‡).

LLG-3 is a ganglioside isolated from the starfish Linchia laevigata. To clarify the structure-activity relationship of the glycan of LLG-3 toward rat pheochromocytoma PC12 cells in the presence of nerve growth factor, a series of mono- to tetrasaccharide glycan derivatives were chemically synthesized and evaluated in vitro. The methyl group at C8 of the terminal sialic acid residue was crucial for neuritogenic activity, and the terminal trisaccharide moiety was the minimum active motif. Furthermore, the trisaccharide also stimulated neuritogenesis in human neuroblastoma SH-SY5Y cells via mitogen-activated protein kinase (MAPK) signaling. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rapidly induced by adding 1 or 10 nM of the trisaccharide. The ratio of phosphorylated ERK to ERK reached a maximum 5 min after stimulation, and then decreased gradually. However, the trisaccharide did not induce significant Akt phosphorylation. These effects were abolished by pretreatment with the MAPK inhibitor U0126, which inhibits enzymes MEK1 and MEK2. In addition, U0126 inhibited the phosphorylation of ERK 1/2 in response to the trisaccharide dose-dependently. Therefore, we concluded that the trisaccharide promotes neurite extension in SH-SY5Y cells via MAPK/ERK signaling, not Akt signaling.

Total syntheses of disulphated glycosphingolipid SB1a and the related monosulphated SM1a.

Total syntheses of two natural sulphoglycolipids, disulphated glycosphingolipid SB1a and the structurally related monosulphated SM1a, are described. They have common glycan sequences and ceramide moieties and are associated with human epithelial carcinomas. The syntheses featured efficient glycan assembly and the glucosyl ceramide cassette as a versatile building block. The binding of the synthetic sulphoglycolipids by the carcinoma-specific monoclonal antibody AE3 was investigated using carbohydrate microarray technology.