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

J. Q. Fan, M. S. Quesenberry, K. Takegawa, S. Iwahara, A. Kondo, I. Kato, Y. C. Lee

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Abstract

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.

Original languageEnglish
Pages (from-to)17730-17735
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number30
DOIs
Publication statusPublished - 1995
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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