Various partially acetylated sialic acid a-ketosides were obtained without expensive protecting group techniques by using trimethyl orthoacetate or dimethylacetamide dimethyl acetal as acetylating agents or by performing a partial Zemplen de-O-acetylation. Using trimethyl orthoacetate as acetylating agent, we synthesized 9-O-acetylated, 8-O-acetylated, and 8,9-di-O-acetylated sialic acid a-ketoside benzyl ester derivatives. The acetylation with dimethylacetamide dimethy1 acetal yielded 9-O-acetylated, 8-O-acetylated, 4,9-di-0-acetylated, 8,9-di-O-acetylated, and 4,8,9-tri-O-acetylated sialic acid a-ketoside benzyl ester derivatives. The partial Zemplen de-O-acetylation permitted the synthesis of 7,8,9-tri-O-acetylated sialic acid a-ketoside benzyl ester derivatives. After catalytic hydrogenation the 8-O-acetylated derivatives 2e and 3d were not stable and were completely converted into the 9-O-acetylated sialic acid a-thioketoside 2k. The N-acetyl-8,9-di-O-acetylneuraminic acid a-aminophenylthioketoside 21 showed a migration of the acetyl group from position 8 to position 7 in polar solvents, yielding a mixture of the 8,9-di-0-and the 7,9-di-O-acetylated derivatives 21 and 2 m in a molar ratio of approximately 1:l. The 7,9-di-O-acetylated derivative 2 m could be separated and was completely stable in methanol. Using the isopropylidene protecting group we could also prepare the 4-O-acetylated sialic acid a-thioketoside 20. Treatment of the 9-O-acetylated derivative 2k with fluorescein isothiocyanate gave the fluorescent derivative 5 .Sialic acids are, as constituents of sialoglycoproteins and gangliosides, widely distributed in the In addition to N-acetylneuraminic acid, a great number of substituted neuraminic acid derivatives occur bearing e.g. acetyl groups at the hydroxyl The acetyl ester groups can be located in the positions 4, 7, 8, or 9 in various combinations. These glycoconjugates containing O-acetylated sialic acids are involved in many biological functions [4]. Especially the O-acetylation is responsible for different biological processes. An acetyl ester group in position 4 blocks the action of bacterial sialidases completely [5]. Acetylation in the glycerol side chain diminishes the sialidase actionL6] and influences in this way the sialic acid catabolism.There are few synthetic methods for obtaining selectively acetylated sialic acid derivatives. First, Haverkamp et al. realized the synthesis of 9-O-acetylated, 4,9-di-O-acetylated and 4,8,9-tri-O-acetylated sialic acid derivatives by using Nacetylimidazole as acetylating agentr71. The selective 9-0-acetylation with trimethyl orthoacetate was described by Ogura et al. [8,91. Also Hasegawa et a1.[l01 synthesized successfully 9-0-and 4,9-di-O-acetylated sialic acid derivatives by use of acetyl chloride/pyridine at -40°C. Recently, a further selective 9-O-acetylation method using dimethylacetamide dimethyl acetal was described byIn addition, Ogura, Hasegawa and coworkers synthesized 4-O-acetylated, 7-O-acetylated, 7,8-di-O-acetylated, 7,9-di-O-acetylate...
2-a-(N-Dansyl-4-aminophenylthio)-./Vacetyl-9-O-acetylneuraminic acid (10) was prepared as a new specific and highly sensitively detectable sialate-9-O-acetyl-esterase substrate. It is built up from a sialidase-stable aminophenyl-a-thioketoside of Nacetylneuraminic acid. By labeling this thioketoside with dansyl chloride a fluorescent neuraminic acid derivative was prepared which allows determinations down to the picomol range. Regioselective acetylation with trimethylorthoacetate results in the corresponding 9-0-acetyl derivative. After incubation with esterase from bovine brain the hydrolysis products were separated on a HPLC column and fluorimetrically detected at 334 nm excitation and 564 nm emission.The K m value of 2.5mM was in the range between the values of the completely unspecific methylumbelliferyl acetate and the less sensitively detectable A r -acetyl-9-O-acetylneuraminic acid which have been used up to now as standard substrates.Zusammenfassung: 2-a-(7V-Dansyl-4-aminophenylthio)-./V-acetyl-9-O-acetylneuraminsäure (10) wurde als neues spezifisches Sialinsäure-9-O-acetylesteraseSubstrat synthetisiert, das noch bis in den PicomolBereich nachgewiesen werden kann. Als Ausgangsverbindung diente das a-Aminophenyl-thioglycosid des Peracetylneuraminsäure-benzylesters. Die Reaktion des Thioglycosids mit Dansylchlorid ergibt ein fluoreszierendes Neuraminsäureglycosid, das durch regioselektive Acetylierung mitTrimethylorthoacetat zum entsprechenden 9-O-acetylierten Neuraminsäure-Derivat umgesetzt wird. Nach Inkubation mit Esterase aus Rinderhirn konnten die Hydrolyseprodukte mittels HPLC getrennt und fluorimetrisch bei einer Anregungswellenlänge von 334 nm und einer Emissionswellenlänge von 564 nm delektiert werden. Der Ä: m -Wert beträgt 2.5mM und liegt zwischen dem des Methylumbelliferylacetats und dem der N-Acetyl-9-O-acetylneuraminsäure, die bislang als Standardsubstrate verwendet wurden.
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