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 N alpha-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 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.
Meldal
M
M
Carlsberg Laboratory, Department of Chemistry, Copenhagen, Denmark.
Breddam
K
K
D000595:Amino Acid Sequence; D005453:Fluorescence; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis*; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D010455:Peptides; D062367:ortho-Aminobenzoates; C002744:3-nitrotyrosine; D014443:Tyrosine; D013381:Subtilisins; D010434:Pepsin A; C000219:anthranilamide