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.