Ac-Leu-pNA contains the amino acid leucine (Leu) linked through an N-acetyl (Ac) group to p-nitroanilide (pNA), forming an amide substrate in which the carboxyl functionality is converted to the p-nitroanilide derivative. The molecule bears an anilide aromatic ring substituted with a para-nitro group and retains the leucine side chain, while the peptide-like backbone presents an N-acetylated amino terminus and a terminal p-nitroanilide as the leaving chromophore. Ac-Leu-pNA is used as a chromogenic substrate in analytical and biochemical assays to monitor protease or peptidase activity by following changes associated with cleavage of the amide bond.
CAT No: CP26606
CAS No:19746-40-8
Synonyms/Alias:19746-40-8;N-Acetyl-L-leucine-p-nitroanilide;Ac-Leu-pNA;Acetyl-L-leucine 4-nitroanilide;(2S)-2-acetamido-4-methyl-N-(4-nitrophenyl)pentanamide;(S)-2-Acetamido-4-methyl-N-(4-nitrophenyl)pentanamide;DTXSID60428584;N-Acetyl-L-leucine P-nitroanilide;DTXCID30379418;AKOS015909056;FD21915;FA110696;Pentanamide, 2-(acetylamino)-4-methyl-N-(4-nitrophenyl)-, (2S)-;Pentanamide,2-(acetylamino)-4-methyl-N-(4-nitrophenyl)-,(2S)-;
Ac-Leu-pNA is an N-acetylated leucine-p-nitroanilide substrate commonly used to monitor protease activity in enzyme assays. The molecule couples a leucine residue to the p-nitroanilide chromophore, enabling colorimetric readout upon enzymatic cleavage. In practical workflows, Ac-Leu-pNA is selected for its straightforward spectrophotometric detection and for supporting routine screening of amino-acid-specific or peptide-bond-cleaving enzymes under controlled conditions.
1. Protease Activity Assays
Ac-Leu-pNA is widely used in protease activity measurements where cleavage releases p-nitroaniline, allowing quantification by absorbance. Enzyme researchers and assay developers use this substrate to compare relative activity across conditions such as buffer composition, pH, temperature, inhibitor presence, or enzyme concentration. The leucine-derived recognition element makes Ac-Leu-pNA a convenient choice for leucine-preferring protease panels and for establishing assay baselines in routine kinetic experiments.
2. Inhibitor Screening Studies
Ac-Leu-pNA supports inhibitor evaluation workflows in chemical biology and enzymology, where small molecules, peptide-based inhibitors, or enzyme modulators are tested for their ability to reduce substrate turnover. Assay labs use the chromogenic response to generate dose-response curves and to rank compounds by relative inhibitory effect under standardized conditions. This format is particularly useful in early-stage hit triage because it provides rapid, plate-compatible readouts that integrate cleanly into high-throughput or semi-high-throughput screening pipelines.
3. Enzyme Kinetics And Characterization
Ac-Leu-pNA is used for kinetic characterization of proteases, including determination of initial rates and the establishment of activity profiles across experimental parameters. Researchers apply it to generate time-course data and to assess how enzyme performance changes with substrate concentration, enabling practical kinetic comparisons between enzyme preparations or variants. Because the readout is directly tied to cleavage of the leucine-p-nitroanilide bond, Ac-Leu-pNA is a practical substrate for method development and for routine characterization in enzymology laboratories.
4. Analytical Method Development
Ac-Leu-pNA is also used as a reference substrate for developing and validating colorimetric enzyme assay conditions, including selecting measurement wavelength, optimizing reaction timing, and confirming linear response ranges. Analytical and process-focused labs employ it to standardize assay performance when building internal SOPs for protease monitoring, such as in research-grade screening or formulation-adjacent process development studies. The reproducible chromophore release supports consistent signal generation for assay calibration and troubleshooting when adapting protocols to new enzyme sources or experimental setups.
1. Cell-based adhesion assays for isolation of snake venom’s integrin antagonists
3. Store-operated Ca2+ entry sustains the fertilization Ca2+ signal in pig eggs
If you have any peptide synthesis requirement in mind, please do not hesitate to contact us at . We will endeavor to provide highly satisfying products and services.
Creative Peptides is a trusted CDMO partner specializing in high-quality peptide synthesis, conjugation, and manufacturing under strict cGMP compliance. With advanced technology platforms and a team of experienced scientists, we deliver tailored peptide solutions to support drug discovery, clinical development, and cosmetic innovation worldwide.
From custom peptide synthesis to complex peptide-drug conjugates, we provide flexible, end-to-end services designed to accelerate timelines and ensure regulatory excellence. Our commitment to quality, reliability, and innovation has made us a preferred partner across the pharmaceutical, biotechnology, and personal care industries.
Read More