Chemo-Enzymatic Synthesis of the Galili Epitope Gal(1!3)Gal(1!4)GlcNAc on a Homogeneously Soluble PEG Polymer by a Multi-Enzyme System Nils Brinkmann, a Martine Malissard, b Maud Ramuz, b Ulrike Ro¨mer, c Thomas Schumacher, c Eric G. Berger, b Lothar Elling, c Christian Wandrey a and Andreas Liese a, * a Institute of Biotechnology, Forschungszentrum Juelich GmbH, 52425 Juelich, Germany b Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland c Institute of Enzyme Technology, University of Duesseldorf, 52426 Juelich, Germany Received 5 April 2001; revised 15 June 2001; accepted 5 July 2001 Abstract—The a-Gal trisaccharide Gala(1!3)Galb(1!4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric sup- port (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of b-1,4-galactosyltransferase (EC 2.4.1.38), a-1,3- galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free a-Gal trisaccharide. # 2001 Elsevier Science Ltd. All rights reserved. Oligosaccharides promise to play an important role as drugsinfutureduetotheirroleinbiologicalrecognition processes. 1,2 However, their synthesis is cumbersome and their purification difficult and time consuming. Here, we present a methodology for the synthesis of oligosaccharides on a homogeneously soluble polymeric support.Thefirststepinvolvesthechemicalsynthesisof a monosaccharide on a soluble polymeric support. Elongation of the saccharide follows by stepwise enzy- matic glycosyltransferase reactions. Finally, the product isisolatedbyaidofdiafiltrationofthepolymerenlarged carbohydrate. A soluble support was chosen in order to enable good accessibility of the carbohydrate substrate to the enzymes. Moreover, by use of a soluble polymer support analytical methods are significantly simplified compared to solid-phase synthesis. This technique, which is applicable both to organic as well as to enzy- matic synthesis, is expected to contribute to further large-scale synthesis of oligosaccharides, especially those containing molecular recognition sites. Pre- viously, such an approach using glycosidases in reverse in combination with a polyethylene glycol-supported solution-phase technique was described. 3 However, these transformations suffer from extremely low con- version. Subsequent to the transglycosylation, the poly- mer (polyethylene glycol, PEG) was codistilled with toluene in order to remove water, redissolved in di- chloromethane and then precipitated by use of tert- butyl methyl ether, followed by filtration and recrys- tallization. Use of the more favorable glycosyl- transferases was not possible due to the fact that metal ions, necessary for the use of transferases, prevent completeprecipitation of PEG. 4 Here we demonstrate that the application of a polymer- supported solution-phase technique as described for a chemical synthesis by Krepinsky et al. 5 in combination with ultrafiltration enables the use of glycosyl- transferases yielding quantitative conversion. This was shown in the example of the trisaccharide Gala(1!3)Galb(1!4)GlcNAc (also referred to as Galili epitope). This a-Gal epitope 6 8 is expressed on xenotissues from pigs and is responsible for the hyper- acute rejection in humans. 9 11 The technique presented here not only contributes to large-scale synthesis of free a-Gal epitopes, but the product, for example polymer bound -Gal trisaccharide itself, may be a useful com- pound to prevent hyperacute xenograft rejection, since 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00474-7 Bioorganic & Medicinal Chemistry Letters 11 (2001) 2503–2506 *Corresponding author. Tel.: +49-2461-6044; fax: +49-2461-3870; e-mail: a.liese@fz-juelich.de