Synthesis of Neoglycoconjugates by Transglycosylation with Arthrobacter protophormiae Endo-β-N-acetylglucosaminidase

The transglycosylation activity of endo-beta-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 beta-GlcNAc (67%), 4-methyl-umbelliferyl beta-GlcNAc (66%), p-nitrophenyl beta-GlcNAc (33%), and (GlcNAc-beta-S-CH2CH2CH2)2 (43%) were lower, because their poor solubilities allowed only 0.05 M or lower concentrations in the reaction mixture. A micromole-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 glycopolymer 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 microM Man9GlcNAc2-sugar chain) and liver (I50 = 74.5 nM) than soybean agglutinin.