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Molecules 2004, 9, 427-439 molecules ISSN 1420-3049 The Preparation of Fluorescence-Quenched Probes for Use in the Characterization of Human Factor Xa Substrate Binding Domains Karen M. Bromfield 1, Julia Cianci 2 and Peter J. Duggan 2,3,* 1 Centre for Green Chemistry, Monash University, Clayton, Victoria, 3800. Australia. 2 School of Chemistry, Monash University, Clayton, Victoria, 3800. Australia. 3 CSIRO Molecular Science, Private Bag 10, Clayton South, Vic 3169, Australia; Phone: +61-3-9545 2560; Fax: +61-3-9545 2446. * Author to whom correspondence should be addressed; e-mail peter.duggan@csiro.au Received: 4 March 2004 / Accepted: 15 March 2004 / Published: 31 May 2004 Abstract: The preparation and characterization by LCMS of a library of 55 fluorescence- quenched peptides is described. The peptides bear a terminal anthranilamide fluorophore and a penultimate 2,4-dinitrophenyl-L-lysine quencher, and will be used to probe the substrate binding domain of the human blood coagulation enzyme, factor Xa. Keywords: Solid phase peptide synthesis, parallel synthesis, fluorescence-quenched substrates, protease, factor Xa. Introduction Factor Xa is an arginine-specific serine protease that operates at the convergence of the extrinsic and intrinsic blood coagulation pathways [1]. The prothrombinase complex, which consists of factors Xa and Va, phospholipid and calcium, is responsible for limited proteolysis of prothrombin to give thrombin. These facts, along with factor Xa’s relatively specific physiological role [2], make it a prime target for the development of new sele