A R T I C L E S
Huang et al.
Endo-A from Arthrobactor protophormiae and the Endo-M from
Mucor hiemali, were able to transfer an oligosaccharide en bloc
from either natural N-glycans or synthetic sugar oxazolines to
a GlcNAc-containing moiety to form a new ꢀ-1,4-glycosidic
linkage in a regio- and stereospecific manner, leading to the
synthesis of complex oligosaccharides, N-glycopeptides and
Scheme 1
6
N-glycoproteins. Endo-A is specific for high-mannose or
hybrid-type N-glycans and has been applied for the synthesis
7
of high-mannose-type oligosaccharides and N-glycopeptides,
whereas Endo-M is able to work on three major types (high-
mannose, hybrid, and complex type) of N-glycans and has been
particularly useful for synthesizing complex type N-glycopep-
8
tides. In particular, the recent findings that synthetic oligosac-
charide oxazolines (the mimics of the oxazolinium ion inter-
mediate of the enzymatic reaction) could be used for Endo-A-
catalyzed transglycosylation have significantly expanded the
scope of the chemoenzymatic method for glycopeptide and
refractory to enzymatic hydrolysis due to the slight structural
modification. Moreover, the discovery of several ENGase-based
glycosynthases, including EndoM-N175A; EndoA-N171A, and
EndoA-E173Q that could promote transglycosylation with sugar
oxazolines of natural N-glycans but lack the ability to hydrolyze
the product, has enabled the synthesis of homogeneous glyco-
9-11
glycoprotein synthesis.
It was found that the highly activated
sugar oxazolines corresponding to the truncated or modified
N-glycans could serve as substrates for the Endo-A-catalyzed
transglycosylation, but the ground-state products formed were
1
2-14
proteins carrying full-size natural N-glycans.
Subsequent
(
(
6) (a) Yamamoto, K. J. Biosci. Bioeng. 2001, 92, 493–501. (b) Wang,
L. X. Carbohydr. Res. 2008, 343, 1509–1522.
studies indicate that Endo-A and Endo-M could accommodate
diverse structures in the aglycon portions of GlcNAc- or Glc-
tagged acceptors for transglycosylation, permitting the introduc-
7) (a) Takegawa, K.; Yamaguchi, S.; Kondo, A.; Kato, I.; Iwahara, S.
Biochem. Int. 1991, 25, 829–835. (b) Takegawa, K.; Yamaguchi, S.;
Kondo, A.; Iwamoto, H.; Nakoshi, M.; Kato, I.; Iwahara, S. Biochem.
Int. 1991, 24, 849–855. (c) Fan, J. Q.; Takegawa, K.; Iwahara, S.;
Kondo, A.; Kato, I.; Abeygunawardana, C.; Lee, Y. C. J. Biol. Chem.
tion of N-glycans into a wide range of natural products, unnatural
15,16
peptides, and even polysaccharides.
The relaxed substrate
specificity of Endo-A and Endo-M, together with the powerful
transglycosylation potential of the glycosynthase mutants,
prompted us to examine the possibility to glue multiple
N-glycans to the complex-type GlcNAc2Man3GlcNAc2-Asn
core through tandem enzymatic transglycosylation. We report
in this contribution the chemoenzymatic synthesis of a class of
novel N-glycan clusters containing multiple N-glycan cores, in
which all monosaccharide residues are connected via defined
native glycosidic bonds found in natural N-glycans. Lectin
microarray analysis of the synthetic N-glycan clusters has
revealed unusual lectin-carbohydrate recognition patterns that
were not observed before.
1
995, 270, 17723–17729. (d) Takegawa, K.; Tabuchi, M.; Yamaguchi,
S.; Kondo, A.; Kato, I.; Iwahara, S. J. Biol. Chem. 1995, 270, 3094–
3
099. (e) Wang, L. X.; Fan, J. Q.; Lee, Y. C. Tetrahedron Lett. 1996,
7, 1975–1978. (f) Wang, L. X.; Tang, M.; Suzuki, T.; Kitajima, K.;
3
Inoue, Y.; Inoue, S.; Fan, J. Q.; Lee, Y. C. J. Am. Chem. Soc. 1997,
19, 11137–11146. (g) Deras, I. L.; Takegawa, K.; Kondo, A.; Kato,
1
I.; Lee, Y. C. Bioorg. Med. Chem. Lett. 1998, 8, 1763–1766. (h) Fujita,
K.; Takegawa, K. Biochem. Biophys. Res. Commun. 2001, 282, 678–
6
82. (i) Fujita, K.; Miyamura, T.; Sano, M.; Kato, I.; Takegawa, K.
J. Biosci. Bioeng. 2002, 93, 614–617. (j) Singh, S.; Ni, J.; Wang, L. X.
Bioorg. Med. Chem. Lett. 2003, 13, 327–330. (k) Li, H.; Singh, S.;
Zeng, Y.; Song, H.; Wang, L. X. Bioorg. Med. Chem. Lett. 2005, 15,
8
95–898. (l) Wang, L. X.; Song, H.; Liu, S.; Lu, H.; Jiang, S.; Ni, J.;
Li, H. ChemBioChem 2005, 6, 1068–1074.
(
8) (a) Yamamoto, K.; Kadowaki, S.; Watanabe, J.; Kumagai, H. Biochem.
Biophys. Res. Commun. 1994, 203, 244–252. (b) Haneda, K.; Inazu,
T.; Yamamoto, K.; Kumagai, H.; Nakahara, Y.; Kobata, A. Carbohydr.
Res. 1996, 292, 61–70. (c) Haneda, K.; Inazu, T.; Mizuno, M.; Iguchi,
R.; Yamamoto, K.; Kumagai, H.; Aimoto, S.; Suzuki, H.; Noda, T.
Bioorg. Med. Chem. Lett. 1998, 8, 1303–1306. (d) Mizuno, M.;
Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.; Aimoto, S.;
Yamamoto, K.; Inazu, T. J. Am. Chem. Soc. 1999, 121, 284–290. (e)
Mori, T.; Sekine, Y.; Yamamoto, K.; Okahata, Y. Chem. Commun.
Results and Discussions
Enzymatic Transglycosylation onto the ꢀ-1,2-Linked GlcNAc
Residues in the Asn-Linked GlcNAc2Man3GlcNAc2 Core.
Construction of an array of N-glycan clusters started with the
preparation of a biotinylated biantennary complex type N-glycan
(
Cambodia) 2004, 2692–2693. (f) Yamanoi, T.; Yoshida, N.; Oda,
(Scheme 1). Coupling of glycosylamine 2 and an activated biotin
Y.; Akaike, E.; Tsutsumida, M.; Kobayashi, N.; Osumi, K.; Yamamoto,
tag (3) gave the GlcNAc-LC-biotin (4), which was then used
as an acceptor for enzymatic transglycosylation. We have
previously reported the synthesis of an asialoglycan oxazoline
K.; Fujita, K.; Takahashi, K.; Hattori, K. Bioorg. Med. Chem. Lett.
2005, 15, 1009–1013. (g) Fujita, K.; Yamamoto, K. Biochim. Biophys.
Acta 2006, 1760, 1631–1635. (h) Makimura, Y.; Watanabe, S.; Suzuki,
T.; Suzuki, Y.; Ishida, H.; Kiso, M.; Katayama, T.; Kumagai, H.;
Yamamoto, K. Carbohydr. Res. 2006, 341, 1803–1808. (i) Haneda,
K.; Takeuchi, M.; Tagashira, M.; Inazu, T.; Toma, K.; Isogai, Y.; Hori,
M.; Kobayashi, K.; Takegawa, K.; Yamamoto, K. Carbohydr. Res.
(11) (a) Li, B.; Song, H.; Hauser, S.; Wang, L. X. Org. Lett. 2006, 8, 3081–
3084. (b) Zeng, Y.; Wang, J.; Li, B.; Hauser, S.; Li, H.; Wang, L. X.
Chem.sEur. J. 2006, 12, 3355–3364. (c) Ochiai, H.; Huang, W.;
Wang, L. X. J. Am. Chem. Soc. 2008, 130, 13790–13803.
(12) Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L. X.;
Yamamoto, K. J. Biol. Chem. 2008, 283, 4469–4479.
2
006, 341, 181–190.
(
9) (a) Fujita, M.; Shoda, S.; Haneda, K.; Inazu, T.; Takegawa, K.;
Yamamoto, K. Biochim. Biophys. Acta 2001, 1528, 9–14. (b) Li, H.;
Li, B.; Song, H.; Breydo, L.; Baskakov, I. V.; Wang, L. X. J. Org.
Chem. 2005, 70, 9990–9996. (c) Wei, Y.; Li, C.; Huang, W.; Li, B.;
Strome, S.; Wang, L. X. Biochemistry 2008, 47, 10294–10304. (d)
Rising, T. W.; Claridge, T. D.; Davies, N.; Gamblin, D. P.; Moir,
J. W.; Fairbanks, A. J. Carbohydr. Res. 2006, 341, 1574–1596. (e)
Rising, T. W.; Claridge, T. D.; Moir, J. W.; Fairbanks, A. J.
ChemBioChem 2006, 7, 1177–1180. (f) Rising, T. W.; Heidecke, C. D.;
Moir, J. W.; Ling, Z.; Fairbanks, A. J. Chem.sEur. J. 2008, 14, 6444–
(13) Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto, K.; Zhang, X.;
Wang, L. X. J. Am. Chem. Soc. 2009, 131, 2214–2223.
(14) Heidecke, C. D.; Ling, Z.; Bruce, N. C.; Moir, J. W.; Parsons, T. B.;
Fairbanks, A. J. ChemBioChem 2008, 9, 2045–2051.
(15) Huang, W.; Ochiai, H.; Zhang, X.; Wang, L. X. Carbohydr. Res. 2008,
343, 2903–2913.
(16) (a) Huang, W.; Groothuys, S.; Heredia, A.; Kuijpers, B. H.; Rutjes,
F. P.; van Delft, F. L.; Wang, L. X. ChemBioChem 2009, 10, 1234–
1242. (b) Ochiai, H.; Huang, W.; Wang, L. X. Carbohydr. Res. 2009,
344, 592–598.
6
464.
(
10) Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L. X. J. Am. Chem.
Soc. 2005, 127, 9692–9693.
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7964 J. AM. CHEM. SOC. 9 VOL. 131, NO. 49, 2009