(2S)-2-Amino-4-methyl-N-(4-nitrophenyl)-pentanamide monohydrochloride
[CAS# 16010-98-3]

Identification

• Classification: Biochemical >> Amino acids and their derivatives >> Leucine derivative
• Name: (2S)-2-Amino-4-methyl-N-(4-nitrophenyl)-pentanamide monohydrochloride
• Synonyms: L-Leucine-4-nitroanilide hydrochloride
• Protein Sequence: L
• Molecular Formula: C₁₂H₁₇N₃O₃^.HCl
• Molecular Weight: 287.74
• CAS Registry Number: 16010-98-3
• SMILES: CC(C)C[C@@H](C(=O)NC1=CC=C(C=C1)[N+](=O)[O-])N.Cl

Properties

• Melting point: 279-280 ºC^*

^* Peschke, Wilhelm; DE 1243204 1967.

Safety Data

• Hazard Symbols: GHS07 Warning
• Hazard Statements: H302-H315-H319-H335
• Precautionary Statements: P261-P305+P351+P338

Discovory and Applicatios

(2S)-2-Amino-4-methyl-N-(4-nitrophenyl)pentanamide monohydrochloride is a chiral amino amide salt. Its base compound is derived from leucine: the (2S)-2-amino-4-methylpentanamide backbone is modified by an anilide: the nitrogen of the amide is bound to a 4-nitrophenyl substituent. The monohydrochloride indicates the presence of one equivalent of HCl, forming a stable salt that is more easily handled and more soluble in polar solvents. Its molecular formula is C₁₂H₁₈ClN₃O₃ (depending on salt form), and its molecular weight is approximately 287 g/mol (as the hydrochloride salt).

The compound is known in the literature under the name “L‑Leucine p‑nitroanilide hydrochloride” (or “L‑Leu‑pNA·HCl”). It is used as a biochemical reagent, particularly in enzymology. In enzyme kinetics, p‑nitroanilide (pNA) derivatives of amino acids are common substrates in colorimetric or fluorimetric assays: when the amide bond to p‑nitroaniline is cleaved (for example by peptidases or proteases), the release of p‑nitroaniline yields a chromogenic signal, because p‑nitroaniline is strongly absorbing in the visible range (around 405 nm). This property makes (2S)-2-amino-4-methyl-N-(4-nitrophenyl)pentanamide a useful synthetic substrate for studying leucine‑specific proteases, leucine aminopeptidases, or other enzymes with specificity for leucine or small hydrophobic amino-acid residues.

In terms of synthesis, the compound is typically prepared by coupling (2S)-2-amino-4-methylpentanamide (i.e., L‑leucinamide) with 4-nitroaniline, or via activated derivatives of the amino acid (such as the acid chloride or active ester) reacting with 4‑nitroaniline under controlled conditions. After amide bond formation, the free base can be converted into the hydrochloride salt by treatment with hydrogen chloride (gas or HCl in solvent), crystallizing as the monohydrochloride. The chiral (2S)‑configuration of the leucinamide backbone is maintained, making the molecule optically active; care in synthesis ensures that racemization is avoided, because the stereocenter at C‑2 (the α-carbon) is sensitive under strong coupling conditions.

Functionally, the nitrophenyl moiety is electron-withdrawing, which both activates the amide for enzymatic cleavage (making it more susceptible to nucleophilic attack by enzyme active sites) and stabilizes the leaving group (p‑nitroaniline). The leucine-derived backbone introduces hydrophobic character and mimics natural amino acid substrates. This makes the molecule especially suitable for assays that quantify leucine-peptidase activity or screen inhibitors of leucine‑cleaving enzymes. Because of the hydrochloride salt form, the compound has higher solubility in water and in buffered assay systems, improving its utility in biochemical protocols.

Historically, p-nitroanilide derivatives of amino acids (like L-leucine-p‑nitroanilide) became widespread in enzymology in the mid- to late-20th century. They provided convenient and reliable synthetic substrates for measuring enzyme kinetics, inhibition, and enzyme specificity. Compared to peptide substrates, p‑nitroanilide derivatives are simpler to synthesize, more stable, and more cost-effective for routine use in assays. Over time, their use expanded from in vitro biochemical characterization to high-throughput screening of potential enzyme inhibitors, especially for proteases and peptidases.

Safety and handling: as with many p‑nitroaniline derivatives, the compound should be handled with care, particularly when dealing with its free base. The hydrochloride salt form reduces volatility and improves handling, but standard laboratory precautions apply: use gloves, avoid inhalation, and store in a cool, dry place. In buffer solutions and assays, its solubility and stability should be tested, as changes in pH or ionic strength may affect its behavior.

In research and industrial contexts, (2S)-2-amino-4-methyl-N-(4-nitrophenyl)pentanamide monohydrochloride remains a valuable tool compound. Its well-characterized structure, its chiral center, and its enzyme substrate properties make it a mainstay in enzymology, particularly in the study of leucine-aminopeptidase-like activities or inhibitors. The ability to monitor cleavage by absorbance of p-nitroaniline simplifies assay design and supports both basic biochemical research and drug-discovery screening.

References

Tuppy H, Wiesbauer U, Wintersberger E (1962) Amino acid‑p‑nitroanilide as a substrate for aminopeptidases and other proteolytic enzymes. Hoppe‑Seyler’s Zeitschrift für Physiologische Chemie 329 1 278–288 DOI: 10.1515/bchm2.1962.329.1.278

Wu X, Chen Y, Aloysius H, Hu L (2011) A novel high‑yield synthesis of aminoacyl p‑nitroanilines: Important synthons for protease substrate design. Beilstein Journal of Organic Chemistry 7 1030–1035 DOI: 10.3762/bjoc.7.117

Muñoz‑Vargas M A, Silva‑Bonilla G E, Caro C, et al (2024) Characterization of leucine aminopeptidase activity using L‑Leu‑pNA: a spectrophotometric approach. PeerJ Life & Environment