Synthesis of 4-Methylcoumarin-7-yloxy Tetra-N-acetyl-β-chitotetraoside, a Novel Synthetic Substrate for the Fluorometric Assay of Lysozyme

4-Methylcoumarin-7-yloxy tetra-N-acetyl-β-chitotetraoside (10) was synthesized from chitin in pure form by a novel procedure. After acetolysis of chitin, chitotetraose tetradecaacetate (4) was isolated by Sephadex LH-20 column chromatography. Compound 4 was chlorinated with dry hydrogen chloride to produce tridecaacetyl chitotetraosyl chloride (6). 7-Hydroxy-4-methylcoumarin sodium salt was condensed with 6 under Koenigs-Knorr reaction conditions, and the final product (10) was obtained by de-O-acetylation of condensation product (8) with sodium methoxide. 4-Methylcoumarin-7-yloxy tri-N-acetyl-β-chitotrioside (9) was also synthesized through chitotriose undecaacetate (3) isolated together with 4 in the same chromatography. Compound 10 was used in a fluorometric assay of lysozyme in comparison with 9. The high sensitivity of fluorometric determination of 7-hydroxy-4-methylcoumarin (12) made it possible to determine lysozyme concentration in the microgram range by using this substrate (10). Unlike the assay using Micrococcus lysodeikticus cell powder, lysozyme assay with this synthetic substrate (10) could be performed directly in biological materials.