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Title: Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione
Authors: Hirata, Izaura Yoshico [UNIFESP]
Cezari, Maria Helena Sedenho [UNIFESP]
Boschcov, Paulo [UNIFESP]
Garratt, Richard Charles
Oliva, Glaucius
Ito, Amando Siuiti
Spisni, Alberto
Franzoni, Lorella
Juliano, Luiz [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Univ Parma
Keywords: ortho-aminobenzoyl-proline
peptide synthesis
protease fluorescent substrate
Issue Date: 1-Jan-1998
Publisher: Kluwer Academic Publ
Citation: Letters in Peptide Science. Dordrecht: Kluwer Academic Publ, v. 5, n. 1, p. 19-28, 1998.
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.
ISSN: 0929-5666
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