Kimio Furuhata

Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Synthesis of 9-O-acyl- and 4-O-acetyl-sialic acids☆

Various 9-O-acyl derivatives of N-acetyl- and N-glycoloyl-neuraminic acid, and O-(5-acetamido-3,5-dideoxy-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosylonic acid)-(2----6)-O-beta-D-galactopyranosyl-(1----4)-D-glucopyranose were regioselectively synthesized by use of ortho esters. In addition, 5-acetamido-4-O-acetyl-D-glycero-D-galacto-2-nonulopyranosonic acid was prepared starting from the benzyl and methyl esters of N-acetylneuraminic acid.

Syntheses of 2-O-glycosyl derivatives of N-acetyl-d-neuraminic acid

Abstract Syntheses of N -acetyl- d -neuraminic acid derivatives are reported. Methyl 4,7,8,9-tetra- O -acetyl- N -acetyl-2-chloro-2-deoxy-β- d -neuraminate ( 3 ) was prepared directly from methyl N -acetyl-β- d -neuraminate ( 2 ) in good yield. Koenigs-Knorr reaction of 3 with an excess of methanol gave the methyl α-glycoside of methyl N -acetyl- d -neuraminate ( 4 ). 2,3- O -Isopropylidene- d -ribono-1,4-lactone, 2,3- O -isopropylideneuridine, and 5-fluoro-2,3- O -isopropylideneuridine reacted with 3 to give anomeric mixtures of methyl N -acteyl- d -neuraminate derivatives. The stereochemistry of these compounds was confirmed from n.m.r. and c.d. spectra, and measurements of the rate of hydrolysis of the glycosidic bond.

Conversion of β-d-C-glucopyranosyl phloroacetophenone to a spiroketal compound

Abstract Treatment of β- d -C-glucopyranosyl phloroacetophenone in water in the presence of a catalytic amount of p-TsOH afforded a spiroketal product. This is the first demonstration of ring conversion in aryl C-glycoside. The structure of the product was determined by 1H-1H COSY, HMQC, HMBC, NOESY, and single crystal X-ray analysis of the corresponding acetylated compound.

A new useful conversion method of naltrexone to 14-deoxynaltrexone

A novel synthetic method of 14-deoxynaltrexone (2), a μ-opioid receptor antagonist possessing a new message-structural part of opioid ligands, was established. Naltrexone methyl ether (5) was first converted to its acetal (24), followed by dehydration with thionyl chloride in pyridine to afford 8,14-dehydroderivative (26) of 24. The resulting unsaturated compound (26) was reduced with PtO 2 under hydrogen to give saturated compound (27), which was then acid-hydrolyzed to afford the desired 14-deoxynaltrexone (23) (3-0-methyl of 2) without degradation of the naltrexone skeleton. The total yield from naltrexone (1) to 23 was 86%. Finally, 23 was demethylated to give 14-deoxynaltrexone (2) in 85% yield. This method provides a useful reaction route to give various important intermediates as a message part to synthesize selectives ligands for the opioid receptor subtypes.

Synthesis and evaluation of influenza virus sialidase inhibitory activity of hinokiflavone-sialic acid conjugates

- The known biflavonoid, hinokiflavone (1) was isolated from the leaves of Metasequoia glyptostroboides Hu et Cheng and displayed influenza A and B virus sialidase inhibitory activity. The unnatural glycoconjugate, hinokiflavone-sialic acid (8) was synthesized and exhibited more potent inhibitory activity.

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs.

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949–56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326–31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19–48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60–81 pmol/mg protein in spleen and lung and 6.9–18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.

Studies on sialic acids I. Determination of anomeric configuration of neuraminic acid derivatives by circular dichroism

CD spectra were recorded for methyl α- and β-glycosides of D-neuraminic acid, and the band at the wave-length lower than 200 nm was attributed to the acetamido group. The Cotton effect at higher wave-length around 220 nm arose from the n-πx transition of the carboxyl group. Thus α-linked glycosides snowed a negative band, while β-glycosides gave arise to a positive band.

Novel practical synthesis of Kdn2en and its C-4 nitrogen-modified derivatives

A practical synthesis of Kdn2en and 4-amino-4-deoxy-Kdn2en has been achieved via a key intermediate, methyl 4,5,7,8,9-penta-O-acetyl-2,6-anhydro-3-deoxy-D-glycero-D-galacto-non-2- enonate, which has been prepared from Kdn in three steps in 91% overall yield.