Synthesis of neoglycoconjugates by transglycosylation with Arthrobacter protophormiae endo-β-N-acetylglucosaminidase. Demonstration of a macro- cluster effect for mannose-binding proteins

The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced dramatically by inclusion of organic solvent in the reaction mixture (see accompanying article; Fan, J.-Q., Takegawa, K., Iwahara, S., Kondo, A., Kato, I., Abeygunawardana, C., and Lee, Y. C. (1995) J. Biol. Chem. 270, 17723-17729). This finding was extended to synthesis of important intermediates for preparation of neoglycoconjugates. When 0.2 M GlcNAc-O-(CH₂)₆NH₂, GlcNAc-O-CH₂CH=CH₂, GlcNAc-O-(CH₂)₃-CH=CH₂, GlcNAc-O-(CH₂)₃NHCOCH=CH₂, GlcNAc-S-CH₂CN, GlcNAc-S-(CH2)₃CH₃, or GlcNAc-S-CH₂-CONHCH₂CH(OMe)₂ were used as acceptors in 30% acetone-containing media, the transglycosylation was accomplished with about 80% yield. The transglycosylation yields to benzyl β-GlcNAc (67%), 4-methyl-umbelliferyl β-GlcNAc (66%), p-nitrophenyl β- GlcNAc (33%), and (GlcNAc-β-S-CH₂CH₂CH₂)₂ (43%) were lower, because their poor solubilities allowed only 0.05 M or lower concentrations in the reaction mixture. A micro-mole-scale synthesis of Man₉GlcNAc₂-O-(CH₂)₃- NHCOCH=CH₂ (Man₉GlcNAc₂-NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by ¹H NMR. Man₉GlcNAc₂-NAP was co-polymerized with acrylamide. The ratio of sugar side chain to acrylamide in this glycopolymer was 1:44 and the molecular weight of glycopolymer was estimated to be between 1,500,000 and 2,000,000 by high performance gel filtration chromatography. The glyco-polymer was shown to be a much more efficient inhibitor of binding by recombinant rat mannose binding protein-carbohydrate recognition domains (MBP-CRD) from serum (I₅₀ = 3.5 μM Man₉GlcNAc₂-sugar chain) and liver (I₅₀ = 74.5 nM) than soybean agglutinin.