Synthesis of 1,5-Anhydro-d-fructose derivatives and evaluation of their inflammasome inhibitors.

Synthesis of several 1,5-Anhydro-d-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow-derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.

Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis.

To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis, we first synthesized 3-O-methyl mannose- and 4-O-methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4-O-methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3-O-methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3'- and 5'-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.

A recyclable heavy fluorous tag carrying an allyl alcohol pendant group: design and evaluation toward applications in synthetic carbohydrate chemistry.

Toward applications in synthetic carbohydrate chemistry, we converted our previous acid-resistant heavy fluorous tag [(Rf)3C-CH2-OH, 1] to allyl alcohol derivatives [(Rf)3C-CH2-O-(CH2)n-CH=CH-CH2-OH, 3 (n=1) or 4 (n=3)] by means of olefin cross metathesis. They were then subjected to ß-glycosylation reactions by using a series of glycosyl donors, including glycosyl bromide and trichloroacetimidates. The terminal OH group in 3 and 4 was found to be ß-glycosylated in moderate yield when 2,3,4,6-tetra-O-benzoyl-D-galactosyl trichloroacetimidate was used as the glycosyl donor. Upon a detachment reaction using Pd(PPh3)4, the initial heavy fluorous tag 1 was recovered in high yield (>90%) together with 1-hydroxy sugar, indicating that not only the allyl ether linkage in the glycosides but also the internal di-alkyl ether linkage in 4 be cleaved by the action of the Pd-catalyst enabling long-range olefin transmigration. Potential utility was demonstrated by using the tetra-O-benzoyl-ß-D-galactosylated derivative of 3 in a series of deprotection, protection and glycosylation reactions, which were conductible in high yields without using chromatographic purification process. These findings prompt us to propose a general scheme in which the acid-resistant heavy fluorous compound 1 is applied as a recyclable tag in synthetic carbohydrate chemistry.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin.

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galß1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galß1pMP, GlcNAcα1,3Galß1pMP, GlcNAcα1,6Galß1pMP, and GlcNAcα1,4GlcAß1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galß1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.

Amino disaccharides having an alpha-(1-->4) or a beta-(1-->4) linkage, their synthesis and evaluation as a potential inhibitor for HIV-1 TAR-Tat.

Amino disaccharides having an alpha-(1-->4) linkage or a beta-(1-->4) linkage have been prepared as the mimics of amino glycoside antibiotics and their inhibition for HIV-1 TAR-Tat were examined. The compound having alpha-(1-->4) linkage denotes better inhibition for TAR-Tat than that of the one having beta-(1-->4).

Rapid oligosaccharide synthesis using a fluorous protective group.

The Bfp-OH, a novel fluorous protecting reagent, was able to be easily prepared. The Bfp group was readily introduced to a carbohydrate, removed in high yield, and recyclable after cleavage. The use of the Bfp group made it possible to synthesize a pentasaccharide by minimal column chromatography purification. Each synthetic intermediate was able to be easily purified only by simple fluorous-organic solvent extraction and monitored by TLC, NMR, and MS.

A novel peptide synthesis using fluorous chemistry.

Three new fluorous supports for peptide synthesis, i.e., the trialkoxybenzhydryl-type (6), the Wang-type (7) and the tert-butyl-type support (8), were prepared. A bioactive peptide TRH was easily synthesized by an Fmoc strategy using the benzhydryl-type fluorous support with fluorous chemistry.