Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione

Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione

Author Hirata, Izaura Yoshico Autor UNIFESP Google Scholar
Cezari, Maria Helena Sedenho Autor UNIFESP Google Scholar
Boschcov, Paulo Autor UNIFESP Google Scholar
Garratt, Richard Charles Google Scholar
Oliva, Glaucius Google Scholar
Ito, Amando Siuiti Google Scholar
Spisni, Alberto Google Scholar
Franzoni, Lorella Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Univ Parma
Abstract The ortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. in fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH2, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH2 or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH3)(2), Abz-Pro-Leu-Gly-NH2 or Abz-pyrrolidine. for all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. in conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.
Keywords ortho-aminobenzoyl-proline
peptide synthesis
protease fluorescent substrate
Language English
Date 1998-01-01
Published in Letters in Peptide Science. Dordrecht: Kluwer Academic Publ, v. 5, n. 1, p. 19-28, 1998.
ISSN 0929-5666 (Sherpa/Romeo, impact factor)
Publisher Kluwer Academic Publ
Extent 19-28
Access rights Closed access
Type Article
Web of Science ID WOS:000073431500004

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