Fluorogenic Peptide-Based Substrates for Monitoring Thrombin Activity

• Published in: ChemMedChem Vol. 7; no. 4; pp. 606 - 617
• Main Authors: van Berkel, Sander S., van der Lee, Bas, van Delft, Floris L., Wagenvoord, Rob, Hemker, H. Coenraad, Rutjes, Floris P. J. T.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01-04-2012; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: Amides - chemistry; Coumarins - chemistry; Dipeptides - chemistry; ETP; Factor Xa - analysis; Factor Xa - metabolism; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; fluorogenic substrates; Humans; Hydrolysis; Kinetics; Malonates - chemistry; peptide synthesis; Peptides; Peptides - chemistry; Peptides - metabolism; thrombin; Thrombin - analysis; Thrombin - metabolism; thrombograms; Triazoles - chemistry
• ISSN: 1860-7179; 1860-7187
• DOI: 10.1002/cmdc.201100560

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 (KM and kcat) 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 P2 or P3 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 KM and kcat 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.
An eye on thrombin: A series of fluorogenic peptidyl‐AMC substrates were synthesized and subjected to a biological evaluation to determine their suitability in the thrombin generation test (TGT). Evaluation of the acquired thrombin generation curves showed that several of these substrates have excellent potential for replacement of the substrate in current use.