Enzyme‐aided construction of medium‐sized alditols of complete O‐linked saccharides

Abstract

We have constructed by enzyme‐aided in vitro synthesis a hexasaccharide alditol Galβ1—4GlcNAcβ1—6Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol and shown that it resists the action of endo‐β‐galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAcβ1—3Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo‐β‐galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAcβ1—6Gal and GlcNAcβ1—3Gal units within linear backbone sequences of all known types of oligo‐(N‐acetyllactosamino)glycans.

Purification and Characterization of UDP-GlcNAc:Galβ1–4GlcNAcβ1–3*Galβ1–4Glc(NAc)-R(GlcNAc to *Gal) β1,6N-Acetylglucosaminyltransferase from Hog Small Intestine

“…The one-dimensional 1 H NMR spectrum of the pentasaccharide product (Fig. 4A, Table IV) is identical with the spectrum previously reported for the authentic pentasaccharide 4 (24). In particular, a doublet with the typical chemical shift and coupling constant (4.584 ppm, 8.3 Hz) of the ␤1,6-linked GlcNAc H1 is present in the spectrum of the pentasaccharide product, and the chemical shifts of Gal2 H1 and Gal2 H4 are characteristic to a 3,6-disubstituted Gal, while Gal4 H1 has a chemical shift typical for a terminal galactosyl residue.…”

“…The uncleaved heptasaccharide probably contained the reducingend ManNAc epimer of 7, formed nonenzymatically. We have observed previously similar ManNAc-epimers as contaminants of other enzymatically synthesized polylactosamines (14,24), and have also observed that the trisaccharide GlcNAc␤1-3Gal␤1-4ManNAc is much less susceptible to endo-␤-galactosidase than the corresponding GlcNAc-epimer, GlcNAc␤1-3Gal␤1-4GlcNAc. 2 As estimated from the NMR spectrum of the original heptasaccharide fraction, discussed below, about 10% of the polylactosamines of this fraction is probably present as ManNAc epimers.…”

Biosynthesis of Branched Polylactosaminoglycans

“…Finally, the identity of the pentasaccharide product generated by PA1 cell lysates was confirmed by the 1 H NMR spectrum (Fig. 1E, Table II); the resonances of the structural reporter groups were practically identical with those of authentic glycan 2 (23). Some of these resonances probably would have been different if the GlcNAc branch had been transferred to C-2 or C-4 of the central galactose unit of glycan 1 (24).…”

“…The presence of the cIGnT6 activity in the murine EC cell lysates suggests that the polylactosamine backbones of these cells may also consist of primary linear chains that carry short a From 1 H NMR spectra of authentic tetrasaccharide 1 and pentasaccharide 2 described by Maaheimo et al (23).…”

The Acceptor and Site Specificity of α3-Fucosyltransferase V

“…High pH anion exchange chromatography with pulsed amperometric detection was carried out with a Dionex series 4500i high pressure liquid chromatography system (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-1 column (4 ϫ 259 mm). The column was equilibrated and run with 40 mM NaOH at a flow rate of 1 ml/min as described (41).…”