Amino acids and peptides. XXXV. Facile preparation of p-nitroanilide analogs by the solid-phase method.
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 Nalpha-,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 Nalpha,NG-protected arginine by any of the coupling methods.
Hojo
K
K
Faculty of Pharmaceutical Sciences and High Technology Research Center, Kobe Gakuin University, Kobe, Japan.
Maeda
M
M
Iguchi
S
S
Smith
T
T
Okamoto
H
H
Kawasaki
K
K
D000596:Amino Acids / Q000737:chemistry*; D000814:Aniline Compounds / Q000138:chemical synthesis*; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D010450:Endopeptidases / Q000737:chemistry*; D006868:Hydrolysis; D010455:Peptides / Q000737:chemistry*; D012116:Resins, Plant; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D000814:Aniline Compounds; D004396:Coloring Agents; D010455:Peptides; D012116:Resins, Plant; D010450:Endopeptidases; D014357:Trypsin