Synthesis, Characterization and Bioassay Studies of p-Nitroanilide Derivatives as Potential Chromogenic Substrate for Endotoxin Screening

Abstract

Five p-nitroanilide derivatives namely, tert-butyl (2-((4-nitrophenyl)amino)-2-oxoethyl)carbamate (1), tert-butyl (1-((4-nitrophenyl)amino)-1-oxopropan-2-yl)carbamate (2), tert-butyl (4-methyl-1-((4-nitrophenyl)amino)-1-oxopentan-2-yl)carbamate (3), tert-butyl (1-((2-((4-nitrophenyl)amino)-2-oxoethyl)amino)-1-oxopropan-2-yl)carbamate (4) and tert-butyl (1-((4-(methylthio)-1-((4-nitrophenyl)amino)-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)carbamate (5) have been successfully synthesized from combination of protected amino acid and p-nitroaniline. These compounds were fully characterized by combination of spectroscopic techniques such as Fourier Transform Infrared (FTIR), Ultraviolet-Visible (UV-Vis), 1H and 13C Nuclear Magnetic Resonance (NMR) and elemental analysis. Common bioassay method, Enzyme-linked Immunosorbent Assay (ELISA) was used to analyze the potential of these compounds to act as chromogenic substrate for endotoxin. The results showed that all of the compounds (except compound 5) gave positive response when interacted with endotoxin by converting the clear solution into yellow cloudy solution. This study also shows that compound 3 that associated with leucine moieties resulted rapid response towards endotoxin which by far acts as the most promising potential chromogenic substrate.