Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation.

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-O t Bu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively. Image 1 • Reliable and scalable solution-phase synthesis of the fluorogenic acyl donor substrate Z-Glu(HMC)-Gly-OH was established. • Substrate properties towards TGase 2-catalysed hydrolysis were confirmed. • Suitability of the substrate for the kinetic characterization of irreversible inhibitors is demonstrated. • Investigation of histamine, serotonin and dopamine with Z-Glu(HMC)-Gly-OH reveals their substrate properties towards TGase 2. [ABSTRACT FROM AUTHOR]