4-Methylumbelliferyl esters as fluorogenic substrates for proteases |
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Authors: | CJ Gray CJSJ DSilva J Boukouvalas SA Barker |
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Institution: | Department of Chemistry, University of Birmingham, P.O. Box 363, Birmingham, B15 2TT, UK |
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Abstract: | 4-Methylumbelliferyl esters of amino acid derivatives have been synthesized using the carbodiimide, disulphite and carbonate methods. Of these, the first was shown capable of preparing 2-naphthyl and 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonyl glycine and benzyloxycarbonyl-citrulline but not of benzoyl-NG-nitroarginine. 2-Naphthyl benzoyl-NG-nitroargininate was prepared successfully using di(2-naphthyl)sulphite. Bis(4-methylumbelliferyl)sulphite could not be prepared but 4-methylumbelliferyl benzoyl-NG-nitroargininate was obtained by the use of an equilibrium method using diphenyl sulphite in the presence of 4-methylumbelliferone. A new reagent, phenyl 4-methylumbelliferyl carbonate, was synthesized and used for the preparation of the 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonylglycine and benzoyl-NG-nitroarginine. The 4-methylumbelliferyl esters of benzyloxycarbonylglycine and benzyloxycarbonylcitrulline were shown to be good substrates for the assay of proteases, including chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4). Disadvantages of 4-methylumbelliferyl esters are discussed. |
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Keywords: | Enzyme assay fluorogenic substrates 4-methylumbelliferone phenyl 4-methylumbelliferyl carbonate citrulline proteolytic enzymes |
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