Rhodamine-based compounds as fluorogenic substrates for serine proteinases.
A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.
D055598:Chemical Phenomena; D002621:Chemistry; D003374:Coumarins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D012235:Rhodamines / Q000138:chemical synthesis / Q000378:metabolism*; D012697:Serine Endopeptidases; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014966:Xanthenes / Q000378:metabolism*
C037983:7-(N-benzyloxycarbonylargininamido)-4-methylcoumarin; D003374:Coumarins; D012235:Rhodamines; D014966:Xanthenes; C037981:N-benzyloxycarbonylargininamidorhodamine; D010450:Endopeptidases; D012697:Serine Endopeptidases