Transfer of ganglioside GM3 oligosaccharide from a water soluble polymer to ceramide by ceramide glycanase. A novel approach for the chemical-enzymatic synthesis of glycosphingolipids

Full text of DOI:10.1021/ja971786c

J. Am. Chem. Soc. 1997, 119, 10555-10556
Transfer of Ganglioside GM3 Oligosaccharide from
10555
a Water Soluble Polymer to Ceramide by Ceramide
Glycanase. A Novel Approach for the
Chemical-Enzymatic Synthesis of
Glycosphingolipids
Shin-Ichiro Nishimura* and Kuriko Yamada
Laboratory for Bio-Macromolecular Chemistry
DiVision of Biological Sciences, Graduate School of
Science, Hokkaido UniVersity, Sapporo 060, Japan
ReceiVed June 2, 1997
It has become a popular topic in recent years that cell surface
carbohydrates play central roles in many biological recognition
processes.1 Of particular interest to glycobiology and medicinal
chemistry is easy, versatile, and practical methodology for the
construction of glycoconjugates of higher structural complexity
and in a combinatorial fashion. Enzyme-assisted strategy for
the synthesis of oligosaccharides is recognized as one of the
promising practical alternatives to chemical synthesis because
of highly stereo- and regioselective reactions with no tedious
protection/deprotection steps.2 As part of an ongoing project
on the feasible and efficient methods for enzymatic syntheses
of carbohydrates using water-soluble glycopolymers as primers,3
our attention was then focused on new synthetic technology
for the construction of sphingoglycolipids on a water-soluble
polymer support having a specific linker that can be recognized
by ceramide glycanase.4
Ganglioside GM3 (1)5 was selected as a target for evaluation
of the present synthetic methodology. Although this trisaccha-
ride sequence (RNeuAc2f3âGal1f4âGlc) was synthesized
previously from methyl lactoside and CMP-NeuAc6 by use of
rat liver âGal1f3/4GlcNAc R-2,3-sialyltransferase7 in modest
yield (39%), the advent of new technologies that amplifies
efficacy of enzymatic strategies is still very much in demand.
This communication describes a facile and efficient enzymatic
synthesis of GM3 using water-soluble polymer support. The
described strategy combines (a) application of water-soluble and
amphiphilic glycopolymer in enzymatic glycolipid synthesis,
1
Figure 1. (A) H- and (B) 13C-NMR spectra of compound 8.
(b) synthesis and utilization of a new lactosyl ceramide-mimetic
monomer in polymer-supported glycoconjugate synthesis, (c)
high efficiency in glycosylation reactions by glycosyltransferases
based on “polymeric glycoside-cluster effect” 8 and easy
purification of the polymer bearing oligosaccharides by simple
gel filtration, and (d) versatile and practical procedure for the
transfer of oligosaccharide products from polymer-support to
ceramide by unique transglycosylation activity of ceramide
glycanase.4b
For the purpose of the present technology, polymerizable
lactose derivative 69 was designed to allow simple preparation
of water-soluble polyacrylamide having lactose residues through
a ceramide mimetic linker derived from a readily available
L-serine derivative 3 (Scheme 1). The lactosyl ceramide
(LacCer) mimetic glycopolymer 7 was obtained in 92% yield
by radical copolymerization of monomeric precursor 6 with
acrylamide in deoxygenated water according to the procedure
previously described.10 This primer support was employed for
sialylation reaction using rat liver âGal1f3/4GlcNAc R-2,3-
sialyltransferase in the presence of CMP-NeuAc according to
the previous report.7 The product 8 (quantitatiVe yield) was
conveniently isolated by a simple column of Sephadex G-25
gel, which can be monitored by NMR measurements (Figure
1). Finally, treatment of 8 with leech ceramide glycanase in
the presence of excess of ceramide as an acceptor and
subsequent chromatographic purifications gave ganglioside GM3
(1) in 61% yield.
* Tel: 81-11-706-3807. Fax: 81-11-706-3435. E-mail: nishimura@
polymer.sci.hokudai.ac.jp.
(1) Reviews: (a) Rademacher, T. W.; Parekh, R. B.; Dwek, R. A. Annu.
ReV. Biochem. 1988, 57, 785. (b) Varki, A. Glycobiology 1993, 3, 97. (c)
Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321.
(2) Reviews: (a) Toone, E. J.; Simon, E. S.; Bednarski, M. D. Whitesides,
G. M. Tetrahedron 1989, 45, 5365. (b) David, S.;Auge, C.; Gautheron, C.
AdV. Carbohydr. Chem. Biochem. 1991, 49, 175. (c) Ichikawa, Y.; Look,
G. C.; Wong, C. H. Anal. Biochem. 1992, 202, 215. (d) Wong, C. H. In
Modern Methods in Carbohydrate Synthesis; Khan, S. H., O’Neill, R. A.,
Eds.; Harwood Academic Publishers: Amsterdam, 1996; p 467.
(3) (a) Zehavi, U.; Herchman, M. Carbohydr. Res. 1984, 128, 160. (b)
Zehavi, U.; Herman, M., Schmidt, R. R.; Bar, T. Glycoconjugate J. 1990,
7, 229. (c) Nishimura, S.-I.; Matsuoka, K.; Lee, Y. C. Tetrahedron Lett.
1994, 35, 5657. (d) Yamada, K.; Nishimura, S.-I. Tetrahedron Lett. 1995,
36, 9493.
The polymer-assisted enzymatic process described above
afforded GM3 in three steps with 56% oVerall yield from a
readily aVailable precursor 6, a remarkable improvement in both
ease of synthesis and overall yield compared to those of
chemical synthesis.5 It should also be noted that each step for
enzymatic sugar-elongations of water-soluble polymer primers
can be clearly characterized by NMR spectroscopy. Since large-
scale preparation of both recombinant glycosyltransferases11 and
sugar nucleotides12 are now possible, the present methodology
(4) (a) Zhou, B.; Li, S.-C.; Laine, R. A.; Huang, R. T. C.; Li, Y.-T. J.
Biol. Chem. 1989, 264, 12272. (b) Li, Y.-T.; Carter, B. Z.; Rao, B. N. N.;
Schweingruber, H.; Li, S.-C. J. Biol. Chem. 1991, 266, 10723. Review:
(c) Li, Y.-T.; Li, S.-C. In Neoglycoconjugates. Preparation and Applications;
Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994; p
251.
(5) (a) Sugimoto, M.; Ogawa, T. Glycoconjugate J. 1985, 2, 5. (b)
Numata, M.; Sugimoto, M.; Shibayama, S.; Ogawa, T. Carbohydr. Res.
1988, 174, 73. (c) Murase, T.; Ishida, H.; Kiso, M.; Hasegawa, A.
Carbohydr. Res. 1989, 188, 71. (d) Numata, M.; Sugimoto, M.; Ito, Y.;
Ogawa, T. Carbohydr. Res. 1990, 203, 205. (e) Ito, Y.; Paulson, J. C. J.
Am. Chem. Soc. 1993, 115, 1603.
(8) For polymeric glycoside-cluster effects by synthetic glycopolymers,
see: (a) Nishimura, S.-I.; Furuike, T.; Matsuoka, K.; Maruyama, K.; Nagata,
K.; Kurita, K.; Nishi, N.; Tokura, S. Macromolecules 1994, 27, 4876. (b)
Matsuoka, K.; Nishimura, S.-I. Macromolecules 1995, 28, 2961. (c) Furuike,
T.; Nishi, N.; Tokura, S.; Nishimura, S.-I. Macromolecules 1995, 28, 7241.
For recent reviews of glycopolymers: (d) Kiessling, L. L.; Pohl, N. L. Chem.
Biol. 1996, 3, 71. (e) Roy, R. Trends Glycosci. Glycotech. 1996, 8, 79.
(9) Details available in Supporting Information.
(10) Reviews: (a) Kochetkov, N. K. Pure Appl. Chem. 1984, 56, 923.
(b) Nishimura, S.-I.; Furuike, T.; Matsuoka, K. Methods Enzymol. 1994,
242, 235.
(11) Colley, K. J.; Lee, E. U.; Adler, B.; Browne, J. K.; Paulson, J. C.
J. Biol. Chem. 1989, 264, 17619.
(12) Some recombinant glycosyl transferases and sugar nucleotides are
now obtainable from Calbiochem.
(6) Sabesan, S.; Paulson, J. C. J. Am. Chem. Soc. 1986, 108, 2068.
(7) Weinstein, J.; de Souza-e-Silva, U.; Paulson, J. C. J. Biol. Chem.
1982, 257, 13845.
S0002-7863(97)01786-1 CCC: $14.00 © 1997 American Chemical Society

10556 J. Am. Chem. Soc., Vol. 119, No. 43, 1997
Scheme 1.a Synthesis of LacCer Mimetic Monomer 6
Communications to the Editor
a Reagents and conditions: (a) AgOTf (1.5 equiv), CH2Cl2, -20 °C, 24 h, 48%; (b) i. H2, Pd-c, MeOH, room temperature, 24 h; ii.
HOOC(CH2)5NHCOCHdCH2 (0.91 equiv), EEDQ (1.0 equiv), EtOH-C6H6, 50 °C, 24 h, 78% from 4; (c) MeONa (0.4 equiv), 25 °C, 2 h, >99%.
EEDQ ) N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
Scheme 2.a Preparation of Primer and Enzymatic Synthesis of GM3
a Reagents and conditions: (a) acrylamide (4.0 equiv), TEMED (0.4 equiv), APS (0.16 equiv), DMSO-H2O, 50 °C, 48 h, 92%; (b) CMP-NeuAc
(1.2 equiv), R-2,3-sialyltransferase (0.3 unit), BSA, MnCl2, CIAP (20 unit), 50 mM sodium cacodylate buffer (pH 7.49), 37 °C, 3 days, >99%; (c)
ceramide (4.85 equiv), ceramide glycanase (0.01 unit), Triton CF-54 (1 drop), 50 mM sodium citrate buffer (pH 6.0), 37 °C, 17 h, 61%. TEMED
) N,N,N′,N′-tetramethylethylene-diamine; APS ) ammonium peroxodisulfate; BSA ) bovine serum albumin; CIAP ) carf intestinal alkaline
phosphate.
and Culture and also by Funds from the Sumitomo Foundation, Izumi
Foundation, and Toray Science Foundation.
using LacCer mimetic polymer should be widely applicable both
for the synthesis of various naturally occurring sphingogly-
colipids and combinatorial synthesis of libraries of glycolipids
varying in the lipid portion.
Supporting Information Available: Experimental details and
spectral and analytical data for all new compounds (16 pages). See
any masthead page for ordering and Internet access instructions.
Acknowledgment. This work was supported in part by a Grant-
in-Aid for Scientific Research from the Ministry of Education, Science,
JA971786C