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Bavaro T.,University of Pavia | Filice M.,Institute Catalisis ICP CSIC | Temporini C.,University of Pavia | Tengattini S.,University of Pavia | And 4 more authors.
RSC Advances | Year: 2014

In this paper a series of 2-iminomethoxyethyl mannose-based mono- and disaccharides have been synthesized by a chemoenzymatic approach and used in coupling reactions with ε-amino groups of lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates. In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 蠑 Lys 91 ≅ Lys 31 > Lys 61 ≅ Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out considering several parameters (amino acids surface exposure and pKa, protein flexibility). The in silico evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis. © The Royal Society of Chemistry 2014.

Filice M.,Institute Catalisis ICP CSIC | Marciello M.,CSIC - Institute of Materials Science | Morales M.D.P.,CSIC - Institute of Materials Science | Palomo J.M.,Institute Catalisis ICP CSIC
Chemical Communications | Year: 2013

The straightforward synthesis of novel enzyme-metalNP nanobiohybrids in aqueous medium was developed. These new nanobiohybrids were excellent multivalent catalysts combining both activities in various sets of synthetic reactions even at ultra-low concentrations (ppb amount). © 2013 The Royal Society of Chemistry.

Marciello M.,CSIC - Institute of Materials Science | Filice M.,Institute Catalisis ICP CSIC | Olea D.,Laboratorios San Ignacio Pharma | Velez M.,Institute Catalisis ICP CSIC | And 2 more authors.
Langmuir | Year: 2014

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g-1 chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations. (Graph Presented). © 2014 American Chemical Society.

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