Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

Abstract

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

Development of an 18F-Labeled Irreversible Inhibitor of Transglutaminase 2 as Radiometric Tool for Quantitative Expression Profiling in Cells and Tissues

“…Determination of the value for 7b under classical conditions in the fluorimetric assay was not possible at pH values higher than pH 6.5 due to the pronounced spontaneous hydrolysis of the fluorogenic substrate. 54 Exemplarily, we also demonstrated the selectivity of [ 18 F]7b toward hTGase 2 by testing the kinetics of its interaction with hTGase 6, which is one of the eight human TGase isoenzymes most closely related to TGase 2. 36,55 As shown in Figure 3, the enzyme−inhibitor complex is formed very slowly with a k inact /K I of 7 M −1 s −1 , which is more than 500-fold less than the respective value for TGase 2.…”

Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure–Activity Relationships, and Pharmacokinetic Profiling

“…For the characterization of the N ε -acryloyllysines toward inhibition of hTGase 2 (human transglutaminase 2), a continuous fluorimetric assay method was used that was recently developed by our group. 59 This assay is based on the measurement of an increase in fluorescence over time due to the TGase 2-catalyzed hydrolysis of the water-soluble fluorogenic acyl donor Z-Glu(HMC)-Gly-OH, resulting in the formation of HMC and Z-Glu-Gly-OH. Due to significant spontaneous hydrolysis of Z-Glu(HMC)-Gly-OH at higher pH values, kinetic characterization of inhibitors was performed at pH 6.5 using six different concentrations of inhibitor.…”

Recent Progress in the Development of Transglutaminase 2 (TGase2) Inhibitors

“…Briefly, transamidation reaction releases 7-hydroxycoumarin (7-HC) from ZFBC, which can be monitored by the increase of fluorescence with excitation at 330 nm and emission at 460 nm (Figure d). Recently, a synthetic derivative containing 7-hydroxy-4-methylcoumarin (7-HMC), Z-Glu­(HMC)-Gly-OH, was developed to measure TG2 activity by fluorescence of 7-HMC released from substrate by hydrolyses and aminolyses …”

“…Recently, a synthetic derivative containing 7-hydroxy-4-methylcoumarin (7-HMC), Z-Glu-(HMC)-Gly-OH, was developed to measure TG2 activity by fluorescence of 7-HMC released from substrate by hydrolyses and aminolyses. 76 Enzyme-Coupled Assay. Enzyme-coupled assays include two-step reactions.…”