An Approach to the Precise Chemoenzymatic Synthesis of 13C-Labeled Sialyloligosaccharide on an Intact Glycoprotein: A Novel One-Pot [3-13C]-Labeling Method for Sialic Acid Analogues by Control of the Reversible Aldolase Reaction, Enzymatic Synthesis of [3-13C]-NeuAc-α-(2→3)-[U-13C]-Gal-β-(1→4)-GlcNAc-β- Sequence onto Glycoprotein, and Its Conformational Analysis by Developed NMR Techniques

Abstract

A one-pot enzymatic 13C-labeling method for the 3-position of sialic acid (NeuAc) analogues has been developed using NeuAc aldolase, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and nucleotide pyrophosphatase (NPP). This method consists of two steps, the first of which is degradation to 2-acetamido-2-deoxy-d-mannose (ManNAc) analogues. This degradation reaction was accelerated by a cofactor regeneration system which converts pyruvic acid into lactic acid using LDH, ADH, and β-nicotinamide adenine dinucleotide oxidized form (β-NAD+). The second step is condensation of the ManNAc analogue with [3-13C]-pyruvic acid newly added after decomposition of the cofactor by nucleotide pyrophosphatase which play a role like switch to stop conversion of pyruvic acid into lactic acid. Five different NeuAc analogues have been labeled in good yields using this newly developed one-pot enzymatic procedure. Following conversion of [3-13C]-NeuAc to CMP-[3-13C]-NeuAc, enzymatic synthesis of [3-13C]-NeuAc-α-(2→3)-[U-13C]-Gal-β-(1→4)-GlcNAc-β-x-ovalbumin (x: hybrid type oligosaccharide) 23 and [3-13C]-NeuAc-α-(2→3)-[U-13C]-Gal-β-(1→4)-GlcNAc-β-OMe 26 (sialyl LacNAc) was performed using bovine β-1,4-galactosyltransferase and rat recombinant α-2,3-sialyltransferase. The 1H chemical shifts of all protons in [3-13C]-NeuAc-α-(2→3)-[U-13C]-Gal-β- on a glycoprotein were assigned by 2D HMQC, 1D HSQC-TOCSY, and the herein described 1D and 2D HSQC-TOCSY-NOESY-TOCSY method. More specifically, the 7-, 8-, and 9-protons of NeuAc could be observed by this HSQC-TOCSY-NOESY-TOCSY method even with only a single 13C atom at the 3-position. In addition, 1D and 2D HMQC-NOESY spectra as well as carbon spin−lattice relaxation times (T1) were measured to compare the conformational properties and dynamic behavior of the sialylgalactoside as part of the sialyl LacNAc 26 and when bound to a glycoprotein 23. These analyses suggested that the conformational properties of sialyl LacNAc are similar for both the conjugated and unconjugated forms, and that the torsional angle of the sialyl linkage, i.e., COOH−C2NeuAc−O−C3Gal, is biased toward the anti (−146.7°) conformation. In addition, the flexibility of galactosyl ring when bound to a glycoprotein appears to be significantly restricted by the attachment of NeuAc as compared with unconjugated sialyl LacNAc.