title: Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis.
abstract: This paper describes the immobilization of the neutral protease fromBacillus subtilisand its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model ofB. subtilisprotease N built in silico by using the structure ofStaphylococcus aureusmetalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield.
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy. teodora.bavaro@unipv.it.
authors.forename: Teodora
authors.initials: T
authors.lastname: Bavaro
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy. giulia.cattaneo01@universitadipavia.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: Cattaneo
authors.affiliation: Department of Food, Environmental and Nutritional Sciences-DeFENS, University of Milano, via L. Mangiagalli 25, I-20133 Milano, Italy. immacolata.serra@unimi.it.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Serra
authors.affiliation: Department for Innovation in Biological, Agro-food and Forest Systems, University of Tuscia, via S. Camillo de Lellis, I-01100 Viterbo, Italy. ilaria.be@unitus.it.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Benucci
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy. massimo.pregnolato@unipv.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Pregnolato
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, I-27100 Pavia, Italy. marco.terreni@unipv.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Terreni
mesh_terms: D000107:Acetylation; D000964:Antimetabolites, Antineoplastic / Q000138:chemical synthesis*; D001412:Bacillus subtilis / Q000201:enzymology*; D000069287:Capecitabine / Q000138:chemical synthesis*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006868:Hydrolysis; D009705:Nucleosides / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*
chemical_list: D000964:Antimetabolites, Antineoplastic; D004800:Enzymes, Immobilized; D009705:Nucleosides; D000069287:Capecitabine; D010447:Peptide Hydrolases
keywords: anticancer products; capecitabine; immobilization; nucleosides; protease N; regioselective hydrolysis