RESUMEN
Novel trisubstituted ethylenes, alkoxy ring-substituted isopropyl 2-cyano-3-phenyl-2-propenoates, RPhCH = C(CN)CO2CH(CH3)2 (where R is 2-methoxy, 3-methoxy, 4-methoxy, 2-ethoxy, 3-ethoxy, 4-ethoxy, 4-propoxy, 4-butoxy, 4-hexyloxy) were prepared and copolymerized with styrene. The ethylenes were synthesized by the piperidine catalyzed Knoevenagel condensation of ring-substituted benzaldehydes and isopropyl cyanoacetate, and characterized by CHN analysis, FTIR, 1H and 13C NMR. All the ethylenes were copolymerized with styrene in solution with radical initiation (ABCN) at 70°C. The compositions of the copolymers were calculated from nitrogen analysis and the structures were analyzed by FTIR, 1H and 13C NMR. Decomposition of the copolymers in nitrogen occurred in two steps, first in the 250-500ºC range with residue (2.6-3.9% wt.), which then decomposed in the 500-800ºC range.
RESUMEN
Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (KM = 15 ± 2 µM), WEHD-MCA (KM = 93 ± 19 µM), WEHDG-MCA (KM = 21 ± 6 µM) and WEHDA-MCA (KM = 151 ± 37 µM), where AMC is 7-amino-4-methylcoumarin and MCA is ß-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases.