ANTHRANILAMIDE AND NITROTYROSINE AS A DONOR-ACCEPTOR PAIR IN INTERNALLY QUENCHED FLUORESCENT SUBSTRATES FOR ENDOPEPTIDASES - MULTICOLUMN PEPTIDE-SYNTHESIS OF ENZYME SUBSTRATES FOR SUBTILISIN CARLSBERG AND PEPSIN

The preparations of Nα-Fmoc-3-nitro-l-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 Å between the centers of the chromophores, and substrates with up to 50 Å between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated. © 1991.