BIOTINYLATED ISOCOUMARINS, NEW INHIBITORS AND REAGENTS FOR DETECTION,LOCALIZATION, AND ISOLATION OF SERINE PROTEASES

Eight new biotinylated, mechanism-based isocoumarin serine protease in hibitors have been designed and synthesized to detect, localize, and i solate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy gr oups are inhibitors of various serine proteases including human leukoc yte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of sp acers between the isocoumarin moiety and the biotin moiety enhanced en zyme inhibitory potency and may also promote binding of the enzyme-inh ibitor complex to avidin. The 3-alkoxy groups conferred selectivity to ward different serine proteases with chymotrypsin being inhibited effe ctively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE an d moderate inhibitors of PPE. Full enzymatic activity was regained aft er the immediate addition of hydroxylamine to the inactivated chymotry psin and PPE derivatives, which indicated that a simple acyl enzyme de rivative is formed initially in the inhibition reaction. Egg avidin di d not effect the rate of spontaneous enzyme reactivation rate while st reptavidin accelerated the reactivation reaction. PPE inhibited by lam ino)caproyl]amino]-4-chloro-3-ethoxyisocoumarin (BIC 5) or amino]capro yl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobiliz ed avidin columns. Most of inhibited PPE could be eluted from the mono meric or tetrameric avidin columns but only a portion (40-70 %) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process. Under ideal circumstan ces and formation of a simple acyl enzyme derivative, BICs should be u seful for the isolation of new active serine proteases. Biotinylated i socoumarins should also have wide applicability for the detection, qua ntitation, and histochemical localization of stable biotinylated serin e protease derivatives in a variety of physiological situations.