Scheme 2 Schematic illustration for semisynthesis of DCCH-ConA. The typical structures of the oxidative degradation of mannose are shown.
Research (No. 08CE2005) from the Ministry of Education,
Science, Sports and Culture of Japan.
Notes and references
1 For reviews, see; (a) D. A. Zachrias, G. S. Baird and R. Y. Tsien, Curr.
Opin. Neurobiol., 2000, 10, 416; (b) H. W. Hellinga and J. S. Marvin,
Trends Biotechnol., 1998, 16, 183; (c) K. A. Giuliano and D. L. Taylor,
Trends Biotechnol., 1998, 16, 135.
2 Synthetic chemosensors for saccharides have been actively developed in
recent years. T. D. James, S. Samankumara and S. Shinkai, Angew.
Chem., Int. Ed. Engl., 1996, 35, 1910.
3 (a) J. S. Marvin and H. W. Hellinga, J. Am. Chem. Soc., 1998, 120, 7;
(b) G. Gilardi, G. Mei, N. Rosato, A. F. Agro and A. E. G. Cass, Protein
Eng., 1997, 5, 479; (c) G. Gilardi, L. Q. Zhou, L. Hibbert and A. E. G.
Cass, Anal. Chem., 1994, 66, 3840.
4 A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M.
Ikura and R. Y. Tsien, Nature, 1997, 388, 882.
Fig. 1 (a) UV-vis spectrum of DCCH-ConA; (b) fluorescence titration
curve of DCCH-ConA with Me-a-Man. [DCCH-ConA] = 3 mM, 50 mM
HEPES buffer (pH 7.0), 1 mM MnCl2, 1 mM CaCl2, 0.1 M NaCl at 15
±1 °C, lex = 435 nm; inset: fluorescence spectral change of DCCH-ConA
by the addition of Me-a-Man.
5 (a) J. R. Sydor, C. Herrmann, S. B. H. Kent, R. S. Doody and M.
Engelhard, Proc. Nat. Acad. Sci. USA, 1999, 96, 7865; (b) G. K. Walkup
and B. Imperiali, J. Am. Chem. Soc., 1996, 118, 3053.
6 (a) G. J. Cotton and T. W. Muir, Chem. Biol., 2000, 7, 253; (b) I.
Hamachi, R. Eboshi, J. Watanabe and S. Shinkai, J. Am. Chem. Soc.,
2000, 122, 4530; (c) I. Hamachi, T. Nagase and S. Shinkai, J. Am. Chem.
Soc., 2000, 122, 12065.
Table 1 The binding constants (K) of DCCH-ConA for saccharides
K/M21
Saccharide
DCCH-ConA
Native ConA
7 V. W. Cornish, K. M. Hahn and P. G. Schultz, J. Am. Chem. Soc., 1996,
118, 8150.
8 (a) H. Bittiger and H. P. Schnebli, Concanavalin A as a Tool, John
Wiley and Sons, New York, 1976; (b) H. Lis and N. Sharon, Chem. Rev.,
1998, 98, 637 and references therein.
Me-a-mannoside
Me-a-glucoside
Me-b-glucoside
Me-a-glactoside
1900
1450
520
11 000a
3 000a
b
—
b
570
—
9 E. Smits, J. B. F. N. Engberts, R. M. Kellogg and H. A. Doren, J. Chem.
Soc., Perkin Trans. 1, 1996, 2873.
10 M. Beppu, T. Terao and T. Osawa, J. Biochem., 1975, 78, 1013. The
labeling reagent 1 was identified by FTIR and 1H-NMR.†
11 Y. Shikata, M. Kuwada, Y. Hayashi, A. Hashimoto, A. Koide and N.
Asakawa, Anal. Chim. Acta, 1998, 365, 241.
12 J. H. Naismith, C. Emmerich, J. Habash, S. J. Harrop, J. R. Helliwell,
W. N. Hunter, J. Raftery, A. J. Kalb and J. Yarib, Acta Crystallogr.,
Sect. D, 1994, 50, 847.
13 G. A. Lemieux and C. R. Bertozzi, Trends Biotechnol., 1998, 16,
506.
14 Chemical modification of the aldehyde group produced by the sugar
oxidation with NaIO4 is a traditional method to modify glycoproteins
(e.g. antibody) and RNA with hydrazine derivatives. It has been
reported that the mild oxidation does not affect protein function. See the
following review articles; (a) D. J. O’Shannessy and M. Wilchek, Anal.
Biochem., 1990, 191, 1; (b) D. J. O’Shannessy and R. H. Quarles,
J. Immunol. Methods, 1987, 99, 153.
a Ref. 16. b The binding constants for Me-a-galactoside and Me-b-
glucoside have not been reported because of their low affinity.
rized in Table 1. It is clear that the order of the affinity constants
for various saccharides is the same as that of native Con A (Me-
a-Man > Me-a-Glc > Me-a-Gal, Me-b-Glc), although the
affinity constants are lower than the literature values of native
Con A17 determined by ITC (isothermal titration calorimetry)
measurement. Significantly these results imply that the molec-
ular recognition event occurring in the binding pocket of
DCCH-Con A can be directly transduced by the fluorescence
signal. This is the first step toward the rational design of
fluorescent saccharide biosensors based on lectins. The reduced
selectivity of DCCH-ConA might be due to the partial blocking
of the sugar-binding pocket by the appended fluorophore and/or
the structural disturbance of the binding site by the unnatural
groups. Details are now under investigation in our laboratory.
We believe that this strategy is so general that other
fluorescent biosensors may be produced showing different
saccharide specificities by the simple replacement of the sugar
part of 1 and the usage of the corresponding lectin.
15 M. S. Herrmann, L. M. Richardson, L. M. Setzler and W. D. Behnke,
Biopolymers, 1978, 17, 2107.
16 UV-vis spectrum of DCCH-ConA showed that 0.5 mol of DCCH was
labeled for 1 mol of ConA monomer. This may be due to the incomplete
oxidation of the mannose unit. Although the further oxidation for longer
than 20 h to complete sugar oxidation was attempted, it was
unsuccessful.
17 (a) F. P. Schwarz, K. D. Puri, R. G. Bhat and A. Surolia, J. Biol. Chem.,
1993, 268, 7668; (b) D. K. Mandal, N. Kishore and C. F. Brewer,
Biochemistry, 1994, 33, 1149.
T. N. is a JSPS fellow for Japanese Junior Scientists. This
research was partially supported by a specially promoted area
(Biotargeting, No. 12019258) and a Grant-in-Aid for COE
230
Chem. Commun., 2001, 229–230