Synthese vonω-Carboxyacyl-L-phenylalanin-arylestern und ihre Verwendung als Substrate für Cathepsin G und Chymotrypsin

Abstract

The synthesis of 5-carboxyvaleryl- and 3-carboxypropionyl-L-phenylalanine .beta.-naphthyl ester (Adi-Phe-ONap, Suc-Phe-ONap) and 3-carboxypropionyl-L-phenylalanine p-nitrophenyl ester (Suc-Phe-ONp) is reported. The 2 latter compounds were obtained in good yields by 3-carboxypropionylation of the L-phenylalanine aryl esters with succinic anhydride at pH values below 6 in aqueous organic solutions. The .beta.-naphthyl esters in particular were sensitive substrates for cathepsin G and chymotrypsin. They are not or only slightly hydrolyzed by other proteinases like elastases, kininogenases, e.g., kallikrein, plasmin, thrombin and trypsin. The spontaneous hydrolysis of the .beta.-naphthyl esters is relatively slow below pH 8. .beta.-Naphthol split-off during the enzyme reaction may be conveniently monitored at 328.5 nm (.epsilon. = 1730 M-1 .times. cm-1) or with an at least 15-fold increase in sensitivity in a discontinuous assay after coupling with Fast Garnet at 520 nm (.epsilon. = 34,800 M-1 .times. cm-1). The increase in absorbance is linear with time and proportional to the amount of enzyme up to A328.5 of at least 0.62. Adi-Phe-ONap is preferentially used for cathepsin G (at 328.5 nm 9.2-fold more sensitive than benzoyl-L-tyrosine ethyl ester, Bz-Tyr-OEt), whereas for chymotrypsin Suc-Phe-ONap is more advantageous (4.2-fold increase in sensitivity at 328.5 nm over Bz-Tyr-OEt). The influence of dimethyl sulfoxide and Brij 35 on the activity of cathepsin G and chymotrypsin was investigated using Suc-Phe-ONap as the substrate. The values of Km and kcat were determined for both enzymes and substrates. Because of the relatively high rates of spontaneous hydrolysis above pH 7.0 the use of Sue-Phe-ONp is less advantageous.