Boc-Ala-Ala-Phe-pNA is a chromogenic peptide substrate with a p-nitroaniline leaving group for tracking proteolysis. The tripeptide core provides a defined recognition sequence for catalytic mapping. Researchers use it in kinetic assays, cleavage-site evaluation, and structure-activity correlation. Its Boc-protection enhances synthetic stability and controlled deprotection.
CAT No: R2251
CAS No:70968-20-6
Synonyms/Alias:Boc-Ala-Ala-Phe-pNA;70968-20-6;tert-butyl N-[(2S)-1-[[(2S)-1-[[(2S)-1-(4-nitroanilino)-1-oxo-3-phenylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]carbamate;DTXSID201121964;FB110711;L-Phenylalaninamide, N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-L-alanyl-N-(4-nitrophenyl)-;tert-butyl (S)-1-((S)-1-((S)-1-(4-nitrophenylamino)-1-oxo-3-phenylpropan-2-ylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-ylcarbamate;
Boc-Ala-Ala-Phe-pNA is a synthetic peptide substrate widely utilized in biochemical and enzymology research. Structurally, it consists of a protected tripeptide sequence—N-tert-butyloxycarbonyl-alanine-alanine-phenylalanine—covalently linked to para-nitroanilide (pNA) at the C-terminus. The inclusion of the pNA chromophore enables sensitive spectrophotometric detection upon enzymatic cleavage, making the compound highly valuable for studying protease activity and substrate specificity. Its well-defined peptide sequence and chromogenic reporting group have established it as a versatile tool in mechanistic studies of serine proteases and related enzyme families.
Enzyme Kinetics Assays: Boc-Ala-Ala-Phe-pNA serves as a preferred substrate in quantitative assays for serine proteases, particularly those with chymotrypsin-like activity. Upon enzymatic hydrolysis, the pNA moiety is released, producing a yellow color that can be measured spectrophotometrically at 405 nm. This property allows for continuous, real-time monitoring of enzyme activity, facilitating precise determination of kinetic parameters such as Km and Vmax. The substrate's defined sequence enables researchers to dissect enzyme-substrate interactions and to compare catalytic efficiencies across enzyme variants or experimental conditions.
Protease Specificity Studies: The tripeptide core of this substrate is specifically designed to probe the substrate preferences of endopeptidases, especially those favoring aromatic residues at the P1 position. By employing Boc-Ala-Ala-Phe-pNA in comparative assays, investigators can map the substrate recognition profiles of various serine proteases or engineered enzyme mutants. Such studies are critical for elucidating structure-activity relationships and for guiding the rational design of inhibitors or modified substrates tailored to specific proteolytic targets.
High-Throughput Screening: The chromogenic nature of the pNA leaving group makes this peptide substrate particularly amenable to high-throughput screening formats. Automated platforms can rapidly assess large compound libraries for protease inhibitors or activators by monitoring changes in absorbance. The robust signal and straightforward readout contribute to reproducibility and scalability, supporting drug discovery efforts and functional genomics studies focused on proteolytic enzymes.
Enzyme Inhibitor Characterization: Boc-Ala-Ala-Phe-pNA is frequently employed to evaluate the potency and mechanism of action of candidate protease inhibitors. By monitoring the rate of pNA release in the presence of test compounds, researchers can determine IC50 values, assess competitive versus non-competitive inhibition, and analyze inhibitor kinetics. Such data are essential for prioritizing inhibitor leads in early-stage research and for guiding subsequent chemical optimization.
Peptide Substrate Engineering: The modular structure of this compound makes it a useful reference point in the development and validation of novel chromogenic peptide substrates. Researchers often use it as a benchmark to compare the performance of newly synthesized analogs or to optimize sequence modifications that enhance selectivity or sensitivity for target enzymes. Its reliability and well-established detection chemistry provide a foundation for advancing peptide-based assay technologies and expanding the repertoire of tools available for protease research.
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