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, Kaoru Takegawa, S. Iwahara, A. Kondo, I. Kato, Y. C. Lee

Research output: Contribution to journalArticle

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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-(CH 2 ) 6 NH 2 , GlcNAc-O-CH 2 CH=CH 2 , GlcNAc-O-(CH 2 ) 3 -CH=CH 2 , GlcNAc-O-(CH 2 ) 3 NHCOCH=CH 2 , GlcNAc-S-CH 2 CN, GlcNAc-S-(CH2) 3 CH 3 , or GlcNAc-S-CH 2 -CONHCH 2 CH(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-CH 2 CH 2 CH 2 ) 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 Man 9 GlcNAc 2 -O-(CH 2 ) 3 - NHCOCH=CH 2 (Man 9 GlcNAc 2 -NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by 1 H NMR. Man 9 GlcNAc 2 -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 (I 50 = 3.5 μM Man 9 GlcNAc 2 -sugar chain) and liver (I 50 = 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 - Jan 1 1995
Externally publishedYes

Fingerprint

Mannose-Binding Lectin
Arthrobacter
Acetylglucosaminidase
Macros
Demonstrations
Acrylamide
Sugars
Acetone
Chromatography
Liver
Organic solvents
Fans
Rats
Polymers
Solubility
Gels
Molecular weight
Carbohydrates
Nuclear magnetic resonance
Gel Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Synthesis of neoglycoconjugates by transglycosylation with Arthrobacter protophormiae endo-β-N-acetylglucosaminidase. Demonstration of a macro- cluster effect for mannose-binding proteins. / Fan, J. Q.; Quesenberry, M. S.; Takegawa, Kaoru; Iwahara, S.; Kondo, A.; Kato, I.; Lee, Y. C.

In: Journal of Biological Chemistry, Vol. 270, No. 30, 01.01.1995, p. 17730-17735.

Research output: Contribution to journalArticle

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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-(CH 2 ) 6 NH 2 , GlcNAc-O-CH 2 CH=CH 2 , GlcNAc-O-(CH 2 ) 3 -CH=CH 2 , GlcNAc-O-(CH 2 ) 3 NHCOCH=CH 2 , GlcNAc-S-CH 2 CN, GlcNAc-S-(CH2) 3 CH 3 , or GlcNAc-S-CH 2 -CONHCH 2 CH(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-CH 2 CH 2 CH 2 ) 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 Man 9 GlcNAc 2 -O-(CH 2 ) 3 - NHCOCH=CH 2 (Man 9 GlcNAc 2 -NAP) was accomplished with 90{\%} yield, and the structure of the transglycosylation product was confirmed by 1 H NMR. Man 9 GlcNAc 2 -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 (I 50 = 3.5 μM Man 9 GlcNAc 2 -sugar chain) and liver (I 50 = 74.5 nM) than soybean agglutinin.",
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AU - Fan, J. Q.

AU - Quesenberry, M. S.

AU - Takegawa, Kaoru

AU - Iwahara, S.

AU - Kondo, A.

AU - Kato, I.

AU - Lee, Y. C.

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N2 - 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-(CH 2 ) 6 NH 2 , GlcNAc-O-CH 2 CH=CH 2 , GlcNAc-O-(CH 2 ) 3 -CH=CH 2 , GlcNAc-O-(CH 2 ) 3 NHCOCH=CH 2 , GlcNAc-S-CH 2 CN, GlcNAc-S-(CH2) 3 CH 3 , or GlcNAc-S-CH 2 -CONHCH 2 CH(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-CH 2 CH 2 CH 2 ) 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 Man 9 GlcNAc 2 -O-(CH 2 ) 3 - NHCOCH=CH 2 (Man 9 GlcNAc 2 -NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by 1 H NMR. Man 9 GlcNAc 2 -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 (I 50 = 3.5 μM Man 9 GlcNAc 2 -sugar chain) and liver (I 50 = 74.5 nM) than soybean agglutinin.

AB - 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-(CH 2 ) 6 NH 2 , GlcNAc-O-CH 2 CH=CH 2 , GlcNAc-O-(CH 2 ) 3 -CH=CH 2 , GlcNAc-O-(CH 2 ) 3 NHCOCH=CH 2 , GlcNAc-S-CH 2 CN, GlcNAc-S-(CH2) 3 CH 3 , or GlcNAc-S-CH 2 -CONHCH 2 CH(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-CH 2 CH 2 CH 2 ) 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 Man 9 GlcNAc 2 -O-(CH 2 ) 3 - NHCOCH=CH 2 (Man 9 GlcNAc 2 -NAP) was accomplished with 90% yield, and the structure of the transglycosylation product was confirmed by 1 H NMR. Man 9 GlcNAc 2 -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 (I 50 = 3.5 μM Man 9 GlcNAc 2 -sugar chain) and liver (I 50 = 74.5 nM) than soybean agglutinin.

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