Amino Acids and Peptides. XXXV. Facile Preparation of p-Nitroanilide Analogs by the Solid-Phase Method

• Published in: Chemical & pharmaceutical bulletin Vol. 48; no. 11; pp. 1740 - 1744
• Main Authors: HOJO, Keiko, MAEDA, Mitsuko, IGUCHI, Shin, SMITH, Timothy, OKAMOTO, Hiroshi, KAWASAKI, Koichi
• Format: Journal Article
• Language: English
• Published: TOKYO The Pharmaceutical Society of Japan 01.11.2000; Pharmaceutical Soc Japan; Maruzen; Japan Science and Technology Agency
• Subjects: 5-amino-2-nitrobenzoic acid; Amino Acids - chemistry; Aniline Compounds - chemical synthesis; Chemistry; Chemistry, Medicinal; Chemistry, Multidisciplinary; Chromatography, High Pressure Liquid; Coloring Agents; Endopeptidases - chemistry; enzyme substrate; Exact sciences and technology; Hydrolysis; Life Sciences & Biomedicine; Organic chemistry; p-nitroanilide; Peptides; Peptides - chemistry; Pharmacology & Pharmacy; phosphoryl chloride method; Physical Sciences; Preparations and properties; Resins, Plant; Science & Technology; solid-phase peptide synthesis; Trypsin - chemistry; p-nitroanilide; solid-phase peptide synthesis; phosphoryl chloride method; COUPLING REAGENTS; CONVENIENT; 5-amino-2-nitrobenzoic acid; enzyme substrate; Serine endopeptidases; Peptides; Enzyme; Phosphoryl Chlorides; Substrate; Peptidases; Trypsin; Tetrapeptide; Dipeptides; Hydrolases; Carboxamide; Peptide synthesis; Solid phase; Chemical synthesis
• ISSN: 0009-2363; 1347-5223
• DOI: 10.1248/cpb.48.1740

p-Nitroanilides of amino acids and peptides are widely used as the chromogenic substrates for the determination of the activity of proteolytic enzymes. However, the preparation of a p-nitroanilide is not easy, in part due to the low nucleophilicity of the amino group of p-nitroaniline. A facile preparation of p-nitroanilide analog by the solid-phase method was investigated. 5-Amino-2-nitrobenzoic acid (Anb5, 2) was used instead of p-nitroaniline (pNA) for preparation of p-nitroanilide analogs. Anb5, 2 was introduced on a p-methylbenzhydrylamine resin without protection of the amino group of Anb5, 2 by the 2-(H-benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TBTU) method in the presence of p-dimethylaminopyridine. The coupling reaction of a Nα-, NG-protected arginine with a Anb5, 2-resin was difficult to achieve by common coupling methods (such as the carbodiimide and diphenylphosphoryl azide methods), but the phosphoryl chloride method was relatively successful. Synthetic benzoyl-Arg-Anb5, 2-NH2 and benzoyl-Arg-pNA were hydrolyzed by trypsin and the both reaction mixtures exhibited same spectroscopic characteristics. H-D-Val-Leu-Arg-Anb5, 2-NH2, an analog of human urine kallikrein substrate, was readily prepared by the solid-phase method. H-Arg-Anb5, 2-OH and H-D-Val-Leu-Arg-Anb5, 2-OH were also synthesized on a Wang resin by the solid-phase method. The aqueous solubility of these free-carboxyl materials was better than those of the corresponding amide analogs. 4-Amino-3-nitrobenzoic acid (Anb4, 3) was also introduced on the p-methybenzhydrylamine resin, but the resulting H-Anb4, 3-resin did not react with Nα-, NG-protected arginine by any of the coupling methods.