Sugar nucleotide regeneration beads (superbeads): a versatile tool for the practical synthesis of oligosaccharides.

Xi Chen, Jianwen Fang, Jianbo Zhang, Ziye Liu, Jun Shao, Przemyslaw Kowal, Peter Andreana, and Peng George Wang* Department of Chemistry, Wayne State UniVersity Detroit, Michigan 48202 ReceiVed October 27, 2000 Application of carbohydrates in modern medicine is limited by the high cost of synthesis of most biologically important glycoconjugates. It is generally recognized that glycosyltransferasecatalyzed glycosylation is one of the most practical approaches.1 Glycosyltransferases catalyze the transfer of a specific sugar from its sugar-nucleotide donor to an acceptor with high regioand stereoselectivity. With increasing availability of recombinant glycosyltransferases in recent years, it can be expected that more researchers will use these enzymes to construct different glycoconjugates. The roadblock for practical use of this methodology is the high cost of necessary sugar nucleotides. So far the best solution is either using multiple-microbial fermentation systems based on patented microorganisms developed by Kyowa Hakko Kogyo Co. Ltd.2 or using in vitro multiple-enzyme sugar nucleotide regeneration systems that avoid the need for stoichiometric amounts of sugar nucleotides. Such cycles were first demonstrated by Wong3 and Whitesides and have been extensively developed by Wong and other groups to produce oligosaccharides.4 Here we report an approach that transfers in vitro multiple enzyme sugar nucleotide regeneration systems onto solid beads (the superbeads) which can be used and reused as common synthetic reagents for production of glycoconjugates.5 The preparation of such sugar nucleotide regeneration beads involves (i) cloning and overexpression of individual N-terminal His6-tagged enzymes along the sugar nucleotide biosynthetic pathway and (ii) co-immobilizing these enzymes onto nickelnitrilotriacetate (NTA) beads.6 The sugar nucleotide regeneration superbeads can then be conveniently combined with glycosyltransferases for specific oligosaccharide sequences. The first generation of superbeads we developed is for UDPGal regeneration. As shown in Scheme 1, the regeneration of UDP-Gal from UDP (byproduct of the galactosylation) requires four enzymes: galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalPUT), glucose-1-phosphate uridylyltransferase (GalU), and pyruvate kinase (PykF). Thus, corresponding galK, galT, galU, and pykF genes were individually amplified from E. coli K-12 genome by polymerase chain reaction, and then inserted into the pET15b vector with a sequence coding for a N-terminal His6-tag, respectively. The enzymes were expressed in E. coli BL21 (DE3) with isopropyl-1-thio-â-D-galactopyranoside (IPTG) induction. Cell lysate mixture with an equal activity of individual enzymes was prepared by combining the cell lysate (in 20 mM Tris-HCl, pH 8.5 buffer containing 1% Triton X-100, 200 μg/mL of lysozyme, and 2 μg/mL of Dnase I) with a relative volume ratio of GalK:GalPUT:GalU:PkyF ) 4:1:1:2.7 The UDPGal regeneration beads were obtained by incubating the cell lysate mixture with Ni2+-NTA resins (3 mL of lysate mixture mL of beads) for 20 min and washing with a Tris-HCl (20 mM, pH 8.0) containing 0.5 M NaCl (Scheme 2). Enzymatic assays indicated that each enzyme was quantitatively immobilized onto the beadswith1.5Uofeach immobilizedenzymepermilliliterofbeads. The application of UDP-Gal regeneration beads was demonstrated by the production of GalR1,3Galâ1,4GlcOBn 1 (Table 1, entries 1 and 2) with a truncated bovine R-1,3-galactosyltransferase (R1,3GalT) expressed in E. coli.8 Oligosaccharides with a terminal GalR1,3Gal sequence (R-Gal epitopes) are desirable as antigens for preventing hyperacute rejection in pig-to-human xenotransplantation.9 In the gram-scale synthesis, the superbeads (40 mL, containing 60 U of GalK, GalPUT, GalU, and PykF) were incubated with a cell lysate of R1,3GalT (40 mL, 40 U, quantitative immobilization), washed by Tris-HCl buffer (20 mM, pH 8.0) containing 0.5 M NaCl, and added to a reaction mixture of LacOBn (1 g, 2.4 mmol), ATP (132 mg, 240 μmol), PEP (912 mg, 4.8 mmol), UDP (100 mg, 240 μmol), Glc-1-P (73 mg, 240 μmol), Gal (540 mg, 3 mmol), MgCl2 (10 mM), MnCl2 (10 mM), and KCl (100 mM) in HEPES buffer (100 mM, pH 7.5) to a total volume of 250 mL. The reaction was stirred at room * To whom correspondence should be addressed. Phone: 313-993-6759. Fax: 313-577-2554. E-mail: [email protected]. (1) (a) Wymer, N.; Toone, E. J. Curr. Opin. Chem. Biol. 2000, 4, 110. (b) Palcic, M. M. Curr. Opin. Biotechnol. 1999, 10, 616. (c) Crout, D. H. G.; Vic, G. Curr. Opin. Chem. Biol. 1998, 2, 98. (2) (a) Tabata, K.; Koizumi, S.; Endo, T.; Ozaki, A. Biotech. Lett. 2000, 22, 479. (b) Endo, T.; Koizumi, S.; Tabata, K.; Ozaki, A. Appl. Microbiol. Biotechnol. 2000, 53, 257. (c) Endo, T.; Koizumi, S.; Tabata, K.; Kakita, S.; Ozaki, A. Carbohydr. Res. 1999, 316, 179. (d) Koizumi, S.; Endo, T.; Tabata, K.; Ozaki, A. Nature Biotechnol. 1998, 16, 847. (3) Wong, C.-H.; Haynie, S. L.; Whitesides, G. M. J. Org. Chem. 1982, 47, 5418. (4) (a) Gilbert, M.; Bayer, R.; Cunningham, A. M.; DeFrees, S.; Gao, Y.; Watson, D. C.; Young, N. M.; Wakarchuk, W. W. Nat. Biotechnol. 1998, 16, 769. (b) Noguchi, T.; Shiba, T. Biosci., Biotechnol., Biochem. 1998, 62, 1594. (c) Zervosen, A.; Elling, L. J. Am. Chem. Soc. 1996, 118, 1836. (d) Look, G. C.; Ichikawa, Y.; Shen, G.-J.; Cheng, G.-J.; Wong, C.-H. J. Org. Chem. 1993, 58, 4326. (e) Wang, P.; Shen, G.-J.; Wang, Y.-F.; Ichikawa, Y.; Wong, C.-H. J. Org. Chem. 1993, 58, 3985. (f) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.; Garcia-Junceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.; Paulson, J. C.; Wong, C.-H. J. Am. Chem. Soc. 1992, 114, 9283. (g) Wong, C.-H.; Wang, R.; Ichikawa, Y. J. Org. Chem. 1992, 57, 4343. (h) Gygax, D.; Spies, P.; Winkler, T.; Pfarr, U. Tetrahedron 1991, 28, 5119. (i) Ichikawa, Y.; Shen, G.-J.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 4698. (5) Representative references on using immobilized enzymes in carbohydrate synthesis: (a) Fujita, K.; Tanaka, N.; Sano, M.; Kato, I.; Asada, Y.; Takegawa, K. Biochem. Biophys. Res. Commun. 2000, 267, 134. (b) Revers, L.; Bill, R. M.; Wilson, I. B.; Watt, G. M.; Flitsch, S. L. Biochim. Biophys. Acta 1999, 1428, 88. (c) Lubineau, A.; Basset-Carpentier, K.; Auge C. Carbohydr. Res. 1997, 300, 161. (d) Asano, N.; Oseki, K.; Kaneko, E.; Matsui, K. Carbohydr. Res. 1994, 258, 255. (e) Roth, S. U.S. Patent 5 583 042, 1996. (f) Roth, S. U.S. Patent 5 288 637, 1994. (g) Roth, S. U.S. Patent 1 580 674, 1993. (6) Ni2+-NTA agarose was from Qiagen, Santa Clarita, CA. Bead size, 45-165 mm; bead structure, cross-linked 6% agarose; support, sepharose CL6B; protein capacity, 300-500 nmol/mL. (7) The activities and specific activities of recombinant enzymes in the cell lysate (25 mL of lysate per 1 L of cell culture) were 25 U/L and 1 U/mg, 100 U/L and 4 U/mg, 100 U/L and 6 U/mg, and 50 U/L and 1 U/mg for GalK, GalPUT, GalU, and PykF, respectively. One unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of product per minute at 24 °C. The amounts of the enzymes were determined by the Lowry method (Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265) after purification. Scheme 1. Biosynthetic Pathway for Galactosides with Regeneration of UDP-Gala