2438
C. D. Heidecke et al. / Carbohydrate Research 344 (2009) 2433–2438
Kondo, A.; Kato, I.; Iwahara, S. Biochem. Int. 1991, 25, 829–835; (c) Takegawa,
K.; Tabuchi, M.; Yamaguchi, S.; Kondo, A.; Kato, I.; Iwahara, S. J. Biol. Chem.
1995, 270, 3094–3099; (d) Takegawa, K.; Yamabe, K.; Fujita, K.; Tabuchi, M.;
Mita, M.; Izu, H.; Watanabe, A.; Asada, Y.; Sano, M.; Kondo, A.; Kato, I.; Iwahara,
S. Arch. Biochem. Biophys. 1997, 338, 22–28; (e) Fujita, K.; Takegawa, K. Biochem.
Biophys. Res. Commun. 2001, 283, 680–686.
assayed with RNase B as the substrate, applying the unit definition
of Endo H activity provided by New England Biolabs. One unit of
Endo A is defined as the amount of enzyme required to remove
>95% of the carbohydrate from 10
tal reaction volume of 10 L in 1 h at 37 °C. Endo M activity was as-
signed as supplied by Professor Yamamoto; 1 unit of Endo M is
defined as the amount of enzyme yielding 1 mol of GlcNAc-
lg of denatured RNase B in a to-
l
5. For some leading references on synthetic applications of Endo A see: (a) Fan, J.-Q.;
Quesenberry, M. S.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.; Lee, Y. C. J. Biol.
Chem. 1995, 270, 17730–17735; (b) Fan, J.-Q.; Huynh, L. H.; Reinhold, B. B.;
Reinhold, V. N.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.; Lee, Y. C.
Glycoconjugate J. 1996, 13, 643–652; (c) Takegawa, K.; Fujita, K.; Fan, J.-Q.;
Tabuchi, M.; Tanaka, N.; Kondo, A.; Iwamoto, H.; Kato, I.; Lee, Y. C.; Iwahara, S.
Anal. Biochem. 1998, 257, 218–223; (d) Fujita, K.; Tanaka, N.; Sano, M.; Kato, I.;
Asada, Y.; Takegawa, K. Biochem. Biophys. Res. Commun. 2000, 267, 134–138.
6. (a) Yamamoto, K.; Fujimori, K.; Haneda, K.; Mizuno, M.; Inazu, T.; Kumagai, H.
Carbohydr. Res. 1997, 305, 415–422; (b) 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; (c) Yamanoi, T.; Tsutsumida, M.; Oda, Y.;
Akaike, E.; Osumi, K.; Yamamoto, K.; Fujita, K. Carbohydr. Res. 2004, 339, 1403–
1406; (d) Osumi, K.; Makino, Y.; Akaike, E.; Yamanoi, T.; Mizuno, M.; Noguchi,
M.; Inazu, T.; Yamamoto, K.; Fujita, K. Carbohydr. Res. 2004, 339, 2633–2635; (e)
Yamanoi, T.; Yoshida, N.; Oda, Y.; Akaike, E.; Tsutsumida, M.; Kobayashi, N.;
Osumi, K.; Yamamoto, K.; Fujita, K.; Takahashi, K.; Hattori, K. Bioorg. Med. Chem.
Lett. 2005, 15, 1009–1013; (f) Haneda, K.; Takeuchi, M.; Tagashira, M.; Inazu, T.;
Toma, K.; Isogai, Y.; Hori, M.; Kobayashi, K.; Takeuchi, M.; Takegawa, K.;
Yamamoto, K. Carbohydr. Res. 2006, 341, 181–190; (g) Makimura, Y.; Watanabe,
S.; Suzuki, T.; Suzuki, Y.; Ishida, H.; Kiso, M.; Katayama, T.; Kumagai, H.;
Yamamoto, K. Carbohydr. Res. 2006, 341, 1803–1808; (h) Murakami, D.;
Shimada, Y.; Kamiya, S.; Yamazaki, K.; Makimura, Y.; Ito, K.; Minamiura, N.;
Yamamoto, K. Arch. Biochem. Biophys. 2008, 477, 299–304.
l
Asn-DNS from Man6GlcNAc2-Asn-DNS in 1 min at 37 °C.
4.2. Analysis of enzymatic glycosylation
Endohexosaminidase-catalysed glycosylations were monitored
by HPLC using a Hewlett–Packard 1050 HPLC instrument using
Clarity software (version 2.4.1.43) connected to an Agilent 1100
variable wavelength detector at 257 nm wavelength. An analytical
HPLC (Phenomenex Gemini 5
used to monitor the reactions, with 2
l
C-18 column, 250 Â 4.6 mm) was
l
L aliquots taken at appro-
priate time intervals. The column was eluted with 22% MeCN/
H2O. The product yield was determined by integration of the prod-
uct and acceptor peaks.
4.3. Typical procedures for enzymatic glycosylation
4.3.1. Method 1: variation of co-solvent
7. Fujita, M.; Shoda, S.-I.; Haneda, K.; Inazu, T.; Takegawa, K.; Yamamoto, K.
Biochim. Biophys. Acta 2001, 1528, 9–14.
Glycosyl amino acid acceptor 1 (100 lg, 621 nmol) and oxazo-
8. (a) Rising, T. W. D. F.; Claridge, T. D. W.; Davies, N.; Gamblin, D. P.; Moir, J. W.
B.; Fairbanks, A. J. Carbohydr. Res. 2006, 341, 1574–1596; (b) Rising, T. W. D. F.;
Claridge, T. D.; Moir, J. W. B.; Fairbanks, A. J. ChemBioChem 2006, 7, 1177–1180;
(c) Rising, T. W. D. F.; Heidecke, C. D.; Moir, J. W. B.; Ling, Z.; Fairbanks, A. J.
Chem. Eur. J. 2008, 14, 6444–6464; (d) Heidecke, C. D.; Ling, Z.; Bruce, N. C.;
Moir, J. W. B.; Parsons, T. B.; Fairbanks, A. J. ChemBioChem 2008, 9, 2045–2051;
(e) Parsons, T.; Moir, J. W. B.; Fairbanks, A. J. Org. Biomol. Chem. 2009, 7, 3128–
3140.
9. (a) Li, B.; Zeng, Y.; Hauser, S.; Song, H. J.; Wang, L.-X. J. Am. Chem. Soc. 2005, 127,
9692–9693; (b) Li, H.; Li, B.; Song, H.; Breydo, L.; Baskakov, I. V.; Wang, L.-X. J.
Org. Chem. 2005, 70, 9990–9996; (c) Wang, L.-X.; Song, H. J.; Liu, S. W.; Lu, H.;
Jiang, S. B.; Ni, J. H.; Li, H. G. ChemBioChem 2005, 6, 1068–1074; (d) Li, H.; Singh,
S.; Zeng, Y.; Song, H.; Wang, L.-X. Bioorg. Med. Chem. Lett. 2005, 15, 895–898; (e)
Zeng, Y.; Wang, J. S.; Li, B.; Hauser, S.; Li, H. G.; Wang, L.-X. Chem. Eur. J. 2006,
12, 3355–3364; (f) Li, B.; Song, H.; Hauser, S.; Wang, L.-X. Org. Lett. 2006, 8,
3081–3084; (g) Umekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang,
L.-X.; Yamamoto, K. J. Biol. Chem. 2008, 283, 4469–4479; (h) Wang, L.-X.
Carbohydr. Res. 2008, 343, 1509–1522; (i) Huang, W.; Ochiai, H.; Zhang, X.;
Wang, L.-X. Carbohydr. Res. 2008, 343, 2903–2913; (j) Wei, Y.; Li, C.; Huang,
W.; Li, B.; Strome, S.; Wang, L.-X. Biochemistry 2008, 47, 10294–10304; (k)
Ochiai, H.; Huang, W.; Wang, L.-X. J. Am. Chem. Soc. 2008, 130, 13790–13803;
(l) Ochiai, H.; Huang, W.; Wang, L.-X. Carbohydr. Res. 2009, 344, 592–598; (m)
Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto, K.; Zhang, X.; Wang, L.-X. J.
Am. Chem. Soc. 2009, 131, 2214–2223.
line donor 2 (3 equiv) were dissolved in a mixture of the appropri-
ate organic solvent (HPLC grade), water and sodium phosphate
buffer (0.4 M solution, pH 6.5, total reaction volume: 50
buffer concentration 0.1 M). Endo M (10 mU) or Endo A (50 mU) or
mutant enzyme (5 g) was added and the temperature was main-
lL; overall
l
tained at 23 °C. Reaction progress was monitored by HPLC analysis
and the product yield was determined by integration of the prod-
uct and acceptor peaks.
4.3.2. Method 2: variation of reaction pH
Glycosyl amino acid acceptor 1 (100
donor (2 or 4, 3 equiv) were dissolved in sodium phosphate buffer
(0.1 M solution of appropriate pH, total reaction volume: 50 L).
Endo M (10 mU) or Endo A (50 mU) or mutant enzyme (5 g) was
lg, 621 nmol) and oxazoline
l
l
added and the temperature was maintained at 23 °C. Reaction pro-
gress was monitored by HPLC analysis and the product yield was
determined by integration of the product and acceptor peaks.
Acknowledgements
10. (a) Mackenzie, L. F.; Wang, Q. R.; Warren, R. A. J.; Withers, S. G. J. Am. Chem. Soc.
1998, 120, 5583–5584; (b) Malet, C.; Planas, A. FEBS Lett. 1998, 440, 208–212.
11. Yin, J.; Li, L.; Shaw, N.; Li, Y.; Song, J. K.; Zhang, W.; Xia, C.; Zhang, R.;
Joachimiak, A.; Zhang, H.-C.; Wang, L.-X.; Liu, Z.-J.; Wang, P. PLoS ONE 2009, 4,
e4658.
12. PDB code 2vtf. See: Suits, M. D. L.; Ling, Z.; Bingham, R. J.; Bruce, N. C.;
Davies, G. J.; Fairbanks, A. J.; Moir, J. W. B.; Taylor, E. J. J. Mol. Biol. 2009,
389, 1–9.
We thank Professor K. Yamamoto (University of Kyoto) for
kindly providing the Endo M enzyme, Professor K. Takegawa (Uni-
versity of Kagawa) for providing the Endo A plasmid, Dr. James
Moir and Dr. Zhenlain Ling (University of York) for production of
WT Endo A and the E173Q and E173H mutants. We also thank
the EPSRC (DTA studentship to TBP) and the BBSRC (Project Grant
BB/D009251/1) for funding.
13. Fan, J.-Q.; Takegawa, K.; Iwahara, S.; Kondo, A.; Kato, I.; Abeyguanawardana, C.;
Lee, Y. C. J. Biol. Chem. 1995, 270, 17723–17729.
14. (a) Akaike, E.; Tsutsumida, M.; Osumi, K.; Fujita, M.; Yamanoi, T.; Yamamoto,
K.; Fujita, K. Carbohydr. Res. 2004, 339, 719–722; (b) Akaike, E.; Yamanoi, T.
Trends Glycosci. Glyc. 2006, 18, 63–71.
15. (a) Synstad, B.; Gaseidnes, S.; Van Aalten, D. M.; Vriend, G.; Nielsen, J. E.;
Eijsink, V. G. Eur. J. Biochem. 2004, 271, 253–262; (b) Williams, S. J.; Mark, B. L.;
Vocadlo, D. J.; James, M. N.; Withers, S. G. J. Biol. Chem. 2002, 277, 40055–
40065; (c) Cetinbas, N.; Macauley, M. S.; Stubbs, K. A.; Drapala, R.; Vocadlo, D. J.
Biochemistry 2006, 45, 3835–3844; (d) Abbott, D. W.; Macauley, M. S.; Vocadlo,
D. J.; Boraston, A. B. J. Biol. Chem. 2009, 284, 11676–11689.
16. Schmidt, B. F.; Ashizawa, E.; Jarnagin, A. S.; Lynn, S.; Noto, G.; Woodhouse, L.;
Estell, D. A.; Lad, P. Arch. Biochem. Biophys. 1994, 311, 350–353.
17. Jha, R.; Davis, J. T. Carbohydr. Res. 1995, 277, 125–134.
18. (a) Yoon, J. H.; Rhee, J. S. Carbohydr. Res. 2000, 327, 377–383; (b) Crout, D. H.; Vic,
G. Curr. Opin. Chem. Biol. 1998, 2, 98–111; (c) Vic, G.; Thomas, D.; Crout, D. H. G.
Enzyme Microb. Tech. 1997, 20, 597–603; (d) Sauerbrei, B.; Thiem, J. Tetrahedron
Lett. 1992, 33, 201–204; (e) Guisan, J. M.; Rodriguez, V.; Soler, G.; Santana, C.;
Fernandez-Lafuente, R.; Bastida, A.; Rosell, C. M.; Lopez, R.; Fernandez-
Mayoralas, A.; Martin-Lomas, M. J. Mol. Catal. 1993, 84, 373–379; (f) Petit,
J.-M.; Paquet, F.; Beau, J.-M. Tetrahedron Lett. 1991, 32, 6125–6128.
References
1. Rich, J. R.; Withers, S. G. Nat. Chem. Biol. 2009, 5, 206–215.
2. (a) Kadowaki, S.; Yamamoto, K.; Fujisaki, M.; Kumagai, H.; Tochikura, T. Agric.
Biol. Chem. 1988, 52, 2387–2389; (b) Kadowaki, S.; Yamamoto, K.; Fujisaki, M.;
Izumi, K.; Tochikura, T.; Yokoyama, T. Agric. Biol. Chem. 1990, 54, 97–106; (c)
Yamamoto, K.; Kadowaki, S.; Watanabe, J.; Kumagai, H. Biochem. Biophys. Res.
Commun. 1994, 203, 244–252; (d) Haneda, K.; Inazu, T.; Yamamoto, K.;
Kumagai, H.; Nakahara, Y.; Kobata, A. Carbohydr. Res. 1996, 292, 61–70; (e)
Yamamoto, K.; Fujimori, K.; Haneda, K.; Mizuno, M.; Inazu, T.; Kumagai, H.
Carbohydr. Res. 1998, 305, 415–422; (f) Mizuno, M.; Haneda, K.; Iguchi, R.;
Muramoto, I.; Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem.
Soc. 1999, 121, 284–290; (g) Fujita, K.; Kobayashi, K.; Iwamatsu, A.; Takeuchi,
M.; Kumagai, H.; Yamamoto, K. Arch. Biochem. Biophys. 2004, 432, 41–49.
3. (a) Takegawa, K.; Nakoshi, M.; Iwahara, S.; Yamamoto, K.; Tochikura, T. Appl.
Environ. Microbiol. 1989, 55, 3107–3112; (b) Takegawa, K.; Yamaguchi, S.;