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ChemMedChem. 2012 Apr;7(4):606-17. doi: 10.1002/cmdc.201100560. Epub 2012 Jan 31.

Fluorogenic peptide-based substrates for monitoring thrombin activity.

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Radboud University Nijmegen, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.


The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (K(M) and k(cat)) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P(2) or P(3) position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K(M) and k(cat) values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et-malonate-Gly-Arg-AMC and N-functionalized Gly-Gly-Arg-AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

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