N-Acetylneuraminic acid and its α2,3/α2,6-glycosidic
linkages with galactose (Neu5Ac-Gal) are major carbohydrate antigen
epitopes expressed in various pathological processes, such as cancer,
influenza, and SARS-CoV-2. We here report a strategy for the synthesis
and binding investigation of molecularly imprinted polymers (MIPs)
toward α2,3 and α2,6 conformations of Neu5Ac-Gal antigens.
Hydrophilic imprinted monoliths were synthesized from melamine monomer
in the presence of four different templates, namely, N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic
acid methyl ester (Neu5Ac-M), 3′-sialyllactose (3SL), and 6′-sialyllactose
(6SL), in a tertiary solvent mixture at temperatures varying from
−20 to +80 °C. The MIPs prepared at cryotemperatures showed
a preferential affinity for the α2,6 linkage sequence of 6SL,
with an imprinting factor of 2.21, whereas the α2,3 linkage
sequence of 3SL resulted in nonspecific binding to the polymer scaffold.
The preferable affinity for the α2,6 conformation of Neu5Ac-Gal
was evident also when challenged by a mixture of other mono- and disaccharides
in an aqueous test mixture. The use of saturation transfer difference
nuclear magnetic resonance (STD-NMR) on suspensions of crushed monoliths
allowed for directional interactions between the α2,3/α2,6
linkage sequences on their corresponding MIPs to be revealed. The
Neu5Ac epitope, containing acetyl and polyalcohol moieties, was the
major contributor to the sequence recognition for Neu5Ac(α2,6)Gal(β1,4)Glc,
whereas contributions from the Gal and Glc segments were substantially
lower.