Selective 13 C-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

Abstract

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of ¹³ C-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the ¹³ C-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.

Keywords: NMR; galectins; molecular recognition; polylactosamine; selective 13C-labels.

Scheme 1

Left. The typical N‐glycan structure containing one poly‐LacNAc chain at the α6 branch. Right. The tri‐LacNAc oligomers containing selective ¹³C‐C₆‐galactose labeling, synthesized and employed in this study.

Figure 1

Synthesis of tri‐LacNAc compounds 5 a, 5 b and 5 c with different ¹³C‐labeling schemes. Reagents and conditions: a) HP‐39, UDP‐GlcNAc; b) LgtB, UDP‐UL‐¹³C₆‐galactose; c) LgtB, UDP‐galactose.

Figure 2

(A) 2D STD‐¹H,¹³C‐HSQC NMR spectra of tri‐LacNAc 5a (with ¹³C₆‐labeled‐galactose at residues A, B and C) in the presence of Gal‐1, Gal‐3 CRD, Gal‐7, Gal‐8 FL, and Gal‐9N (ligand:galectin molar ratios are 30:1). Left, 2D ¹H,¹³C‐HSQC spectrum with signal assignment and right, 2D STD‐¹H,¹³C‐HSQC NMR spectra. (B) Corresponding sum of STD effects (in percent) for all signals of the terminal galactose A (blue) and the two spectrally indistinguishable residues B and C (gray). The STD sum was computed from the net signal integrals in the 2D ¹H,¹³C‐HSQC spectrum with on‐resonant protein saturation (2 s at 0.84 ppm using a train of 33 ms PC9_4 90° pulses) vs. off‐resonant irradiation (at −25 ppm), * with exclusion of the indistinguishable C1‐H1 and C6‐H6 signals, and C5‐H5 in Gal‐8 FL. See the Supplementary Material for further details.

Figure 3

Analysis of 2D STD‐¹H,¹³C‐HSQC NMR experiments for 5b and 5c with the different galectins. The plots represent the STD intensity in % ((cross‐peak integral in the STD spectrum/cross‐peak intensity in the reference spectrum) ×100) of every CH cross‐peak of the galactose residue (represented in the X axis). Pertaining 2D STD‐¹H,¹³C‐HSQC NMR spectra were recorded as detailed in Figure 2.