Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containingortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

✍ Scribed by Izaura Yoshico Hirata; Maria Helena Sedenho Cezari; Clovis Ryuichi Nakaie; Paulo Boschcov; Amando Siuiti Ito; Maria Aparecida Juliano; Luiz Juliano

• Book ID: 104628734
• Publisher: Springer Netherlands
• Year: 1995
• Tongue: English
• Weight: 660 KB
• Volume: 1
• Category: Article
• ISSN: 1573-3149
• DOI: 10.1007/bf00119771

✦ Synopsis

A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N~-Boc or -Fmoc derivative of glutamic acid with the a-carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the a-amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.