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Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Zeng, Steffen ; Gutiérrez Gallego, Ricardo ; Dinter, Andre ; Malissard, Martine ; Kamerling, Johannis ; Vliegenthart, Johannes ; Berger, Eric

In: Glycoconjugate Journal, 1999, vol. 16, no. 9, p. 487-497

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    Titre
    • Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin
    Auteur
    • Zeng, Steffen. Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Gutiérrez Gallego, Ricardo. Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB, Utrecht, The Netherlands
    • Dinter, Andre. Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Malissard, Martine. Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Kamerling, Johannis. Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB, Utrecht, The Netherlands
    • Vliegenthart, Johannes. Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075, NL-3508 TB, Utrecht, The Netherlands
    • Berger, Eric. Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Glycoconjugate Journal, 1999, vol. 16, no. 9, p. 487-497. Kluwer Academic Publishers-Plenum Publishers
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:319871
    Summary
    Sialyl Lewis x (sLex) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-α-D-galactosaminide (GalNAc(α1-pNp as core substrate, the sLex-oligosaccharide Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(β1-3)GalNAc(α1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of β3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 β6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(β1-6)[Gal(β1-3)]GalNAc(α1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk β4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant α3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human α3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 µmol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure