Rapid preparation of a glycan oxazoline and a homogeneously glycosylated antibody with an enzyme-immobilized monolithic column.

Chemo-enzymatic glycan engineering is considered to be one of the most promising strategies to enhance efficiency in pharmaceutical research. However, it is assumed that this technology has limited industrial application for the production of biological therapeutics because of the high cost of the process. In this study, we developed a scheme for rapidly preparing a glycan oxazoline and a homogeneously glycosylated antibody. The enzyme-immobilized monolith and the flow chemistry-based approach enabled a glycan oxazoline and a homogeneously glycosylated antibody to be obtained at the gram scale from starting materials (sialylglycopeptide and heterogeneously glycosylated protein) within 2.5 h. This cost-effective scheme for obtaining a large amount of glycan donors and homogeneously glycosylated proteins in a short time will be helpful to implement glycan engineering technology for industrial purposes such as pharmaceutical production.

Synthesis of a fluorescent probe for measuring the activity of endo-ß-N-acetylglucosaminidases recognizing hybrid-type N-glycans.

A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases) interacting with hybrid-type N-glycans. This was achieved using a dual-labeled fluorescent probe with a nonasaccharide structure. We produced the nonasaccharide skeleton by the stepwise glycosylation of the galactose residue on a galactosyl chitobiose derivative. Next, we introduced azido and acetoxy groups into the nonasaccharide derivative in a stepwise manner, which led to stereochemistry inversion at both the C-4 and C-2 hydroxy groups on its galactose residue. The protecting groups of the resulting nonasaccharide derivative were removed, and the derivative was labeled with an N-methylanthraniloyl group to obtain a reporter dye and a 2,4-dinitrophenyl group as a quenching molecule to obtain target probe 1. The use of this probe along with a microplate reader enabled a facile evaluation of the hydrolytic activities of ENGases Endo-H, Endo-M, Endo-F3, Endo-S, and Endo-CC. Furthermore, this probe could also assist in the search for novel ENGases that are specific to hybrid-type N-glycans.

A silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose functions as an efficient photosensitizer for photodynamic therapy.

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.

Protecting-group-free glycosylation of phosphatidic acid in aqueous media.

The glycosylation of unprotected carbohydrates has emerged as an area of significant interest because it obviates the need for long reaction sequences involving protecting-group manipulations. Herein, we report the one-pot synthesis of anomeric glycosyl phosphates through the condensation of unprotected carbohydrates with phospholipid derivatives while retaining high stereo- and regioselective control. The anomeric center was activated using 2-chloro-1,3-dimethylimidazolinium chloride to facilitate condensation with glycerol-3-phosphate derivatives in an aqueous solution. A water/propionitrile mixture provided superior stereoselectivity while maintaining good yields. Under these optimized conditions, the condensation of stable isotope-labeled glucose with phosphatidic acid provided efficient access to labeled glycophospholipids as an internal standard for mass spectrometry.

Convergent synthesis of oligomannose-type glycans via step-economical construction of branch structures.

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection-deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

Chemical Synthesis of Phosphatidylglucoside.

1-stearoyl (18:0)-2-arachidoyl (20:0)-sn-glycero-3-phospho-ß-D-glucoside (Phosphatidylglucoside or PtdGlc) was synthesized by direct coupling of D-glucose with the phosphate group of phosphatidic acid (18:0, 20:0). Selective in situ activation of the anomeric center of D-glucose by 2-chloro-1,3-dimethylimidazolinium chloride (DMC) in aqueous media allows the omission of protecting groups while furnishing the required ß-phosphate linkage with high selectivity. The described method is suitable to access PtdGlc in mg scale utilizing a simple two step purification protocol.

A fluorogenic probe for core-fucosylated glycan-preferred ENGase.

A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.

Luminescence detection of peptide:N-glycanase activity using engineered split inteins.

A split intein-based method has been developed to detect peptide:N-glycanase (PNGase) activity in live cells. PNGase cleaves the linkage between N,N'-diacetylchitobiose and the Asn side-chain of N-intein peptides and the products react rapidly with C-intein by protein trans-splicing to generate an active luciferase.

Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera.

Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.

Synthesis of 3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside (ODSG) as a lipase-resistant SQAP derivative.

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).

Efficient synthesis of α(1,2)-linked oligomannoside derivatives through one-pot glycosylation.

An α(1,2)-linked oligomannoside derivative having a free C-2 hydroxyl group and a C-3 pivaloyl group was synthesized from a thiophenyl mannose derivative 1 using a one-pot self-condensation and applying a α-stereoselective procedure. The mannosylation exclusively generated α-mannoside linkages. The observed α-directing effect was rationalized by the remote participation of the pivaloyl group in C-3 position. The polymerization degree was controlled by the promoter amount providing the mannobiose derivative as a major product. Applying this method eliminated many synthetic steps. The α(1,2)-linked oligomannoside derivatives, which are key intermediates for the synthesis of oligomannose type N-glycans for glycoproteins, were easily prepared.

A bioactive mammalian disaccharide associated with autoimmunity activates STING-TBK1-dependent immune response.

Glycans from microbial pathogens are well known pathogen-associated molecular patterns that are recognized by the host immunity; however, little is known about whether and how mammalian self-glycans activate the host immune response, especially in the context of autoimmune disease. Using biochemical fractionation and two-dimensional HPLC, we identify an abundant and bioactive free glycan, the Manß1-4GlcNAc disaccharide in TREX1-associated autoimmune diseases. We report that both monosaccharide residues and the ß1-4 linkage are critical for bioactivity of this disaccharide. We also show that Manß1-4GlcNAc is produced by oligosaccharyltransferase hydrolysis of lipid-linked oligosaccharides in the ER lumen, followed by ENGase and mannosidase processing in the cytosol and lysosomes. Furthermore, synthetic Manß1-4GlcNAc disaccharide stimulates a broad immune response in vitro, which is in part dependent on the STING-TBK1 pathway, and enhances antibody response in vivo. Together, our data identify Manß1-4GlcNAc as a novel innate immune modulator associated with chronic autoimmune diseases.

Fluorogenic probe for measuring high-mannose type glycan-specific endo-ß-N-acetylglucosaminidase H activity.

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-ß-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the ß-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.

Fluorescence Quenching-based Assay for Measuring Golgi endo-α-Mannosidase.

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G-EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G-EM activity to be evaluated easily and quantitatively.

A New Fluorogenic Probe for the Detection of endo-ß-N-Acetylglucosaminidase.

We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-ß-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.

A novel glucuronoyl esterase from Aspergillus fumigatus-the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters.

Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.

Specificity of Donor Structures for endo-ß-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions.

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo-ß-N-acetylglucosaminidase from Mucor hiemalis (endo-M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine-linked glycoprotein glycan with a ß-mannose unit of a non-natural-type monosaccharide, including ß-glucose, ß-galactose, and ß-talose in place of the ß-mannose moiety. The transglycosylation activity of wildtype (WT) endo-M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p-nitrophenyl N-acetylglucosaminide (GlcNAc-pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.

Evaluation of Sucrose Fatty Acid Esters as Lubricants in Tablet Manufacturing.

Lubricants are essential additives in tablet formulations. Magnesium stearate (Mg-St) is the most commonly used lubricant in tableting. Here, we used sucrose fatty acid ester (SE) as an additive to manufacture tablets by direct compression. We evaluated the effects of hydrophile-lipophile balance (HLB) and the amount of SE on the flowability of a pharmaceutical powder using angle of repose and practical angle of internal friction measurements. In addition, we investigated the effects of SE on tablet properties. When SEs with an HLB ≥3 were added, the angle of repose was approximately the same as that of a pharmaceutical powder containing Mg-St, with no major differences in flowability. However, the practical angle of internal friction became closer to pharmaceutical powder containing Mg-St as HLB decreased. As HLB increased, the practical angle of internal friction approached the value of additive-free pharmaceutical powder. Tablets containing 2.0% Mg-St had a mean hardness of 40 N and disintegrated in approximately 6 min, whereas tablets containing 2.0% SE (low HLB) had a mean hardness of approximately ≥80 N and disintegrated within 3 min. The results indicate that SEs can be used as lubricants in tablet production by direct compression and to reduce problems associated with the use of Mg-St. In particular, we suggest that SEs with low HLB values can be used as excipients to achieve high tablet hardness and short disintegration time.

Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate.

Enzymatic synthesis and the reverse transcription of RNAs containing 2'-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2'-O-carbamoyl uridine triphosphate (UcmTP). UcmTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection.

Preferential binding of E. coli with type 1 fimbria to D-mannobiose with the Manα1 → 2Man structure.

Manα1 → 2Man, Manα1 → 3Man, Manα1 → 4Man, and Manα1 → 6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco™ beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1 → 2Man1 → beads, Manα1 → 3Man1 → beads, Manα1 → 4Man1 → beads, and Manα1 → 6Man1 → beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1 → 2Man disaccharide.