Boc-LGR-pNA, a chromogenic substrate for horseshoe crab clotting enzyme, which is used in quantitative assays of endotoxin.
CAT No: 10-101-349
CAS No:68223-96-1
Synonyms/Alias:ENDOTOXIN SUBSTRATE;68223-96-1;tert-butyl N-[(2S)-1-[[2-[[(2S)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-4-methyl-1-oxopentan-2-yl]carbamate;SCHEMBL3396260;tert-Butyl ((S)-1-((2-(((S)-5-guanidino-1-((4-nitrophenyl)amino)-1-oxopentan-2-yl)amino)-2-oxoethyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate;
Chemical Name:tert-butyl N-[1-[[2-[[5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]amino]-2-oxoethyl]amino]-4-methyl-1-oxopentan-2-yl]carbamate
Boc-Leu-Gly-Arg-pNA. Acetate is a synthetic peptide substrate widely utilized in biochemical research, particularly in studies involving protease activity and specificity. Structurally, it features a blocked N-terminus (Boc group), a tripeptide sequence (leucine-glycine-arginine), and a para-nitroanilide (pNA) chromogenic group at the C-terminus, supplied as the acetate salt. The incorporation of the pNA moiety enables sensitive colorimetric detection upon enzymatic cleavage, making this compound a valuable tool for investigating serine proteases and related enzymatic processes. Its precise sequence design allows for targeted interaction with specific proteolytic enzymes, facilitating kinetic and mechanistic studies in a range of experimental contexts.
Enzyme activity assays: Boc-Leu-Gly-Arg-pNA. Acetate is extensively employed as a chromogenic substrate in quantitative assays for serine proteases, especially those recognizing arginine at the P1 position, such as trypsin-like enzymes. Upon enzymatic hydrolysis, the release of the p-nitroaniline group yields a measurable yellow color, enabling real-time monitoring of protease activity using spectrophotometric techniques. This property allows researchers to determine enzyme kinetics, substrate specificity, and inhibitor potency with high sensitivity and reproducibility in both purified enzyme systems and complex biological mixtures.
Protease inhibitor screening: The substrate's well-defined cleavage site and chromogenic readout make it an ideal candidate for high-throughput screening of protease inhibitors. By monitoring the inhibition of pNA release in the presence of candidate molecules, investigators can efficiently evaluate the efficacy and selectivity of new inhibitor compounds. This approach supports drug discovery and biochemical tool development by providing rapid, quantitative data on inhibitory activity against target serine proteases.
Substrate specificity profiling: Boc-Leu-Gly-Arg-pNA. Acetate serves as a model substrate for mapping protease substrate preferences and elucidating enzyme-substrate interaction mechanisms. By comparing the hydrolysis rates of this peptide with those of structurally related analogs, researchers can dissect the influence of amino acid sequence and side-chain chemistry on enzyme recognition and catalysis. Such studies contribute to a deeper understanding of protease function, substrate binding determinants, and the molecular basis of enzymatic selectivity.
Biochemical method validation: The use of this chromogenic peptide substrate is integral to the validation and standardization of protease assay protocols in research and quality control laboratories. Its reliable performance and well-characterized cleavage profile allow for the calibration of assay conditions, assessment of enzyme preparation quality, and benchmarking of analytical sensitivity. Employing Boc-Leu-Gly-Arg-pNA. Acetate in these contexts ensures consistency and comparability across experimental workflows, supporting robust and reproducible biochemical analyses.
Enzyme mechanism elucidation: Application of this substrate in mechanistic studies enables detailed exploration of protease catalytic pathways and intermediate formation. By analyzing the kinetics of pNA release under varying conditions, researchers can infer mechanistic parameters such as transition state stabilization, catalytic efficiency, and allosteric modulation. These insights are essential for advancing fundamental knowledge of enzyme function and guiding the rational design of selective protease modulators or engineered enzyme variants for research applications.
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