Donor specificity of Trypanosoma cruzi trans-sialidase (TcTs) has been investigated with modified 2-[4-methylumbelliferone]-alpha- ketoside of N-acetyl-D-neuraminic acid (4MU-NANA) as donor and lactose as acceptor. 4MU-NANA was treated with periodate under mild conditions to generate an aldehyde on the exocyclic side chain. The oxidized 4MU-NANA was derivatized with various primary amines by reductive amination to yield potential donors. High-performance anion-exchange chromatography equipped with pulsed amperometric detector was used to assay the transglycosylation activity of TcTs. Several modified 4MU-NANA derivatives served as substrates by TcTs and they may be utilized to make valuable intermediates, including those for fluorescence energy transfer measurement or photoaffinity labeling experiment.