Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

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 3-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 LCMS
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 [ Lys 91 y Lys 31 > Lys
61 y 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.