A. Bernardi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2197±2200
2199
Figure 1. The eects of ligand binding on the ¯uorescence intensity of Trp88 in CTB. In A CTB (0.5 mM) was titrated with micromolar amounts of
o-GM1 1 (open circles, dashed line) or of mimic 2 (closed circle, solid line), which are shown plotted against the normalized, absolute value of the
relative ¯uorescence intensity (jdIj) measured at the emission maximum of Trp88. In B the same experiment was performed using 0.5 mM CTB and
micromolar concentrations of 4 (open triangles, dashed line), 3 (closed circles, solid line) and 5 (open squares, dashed line) or 2 mM CTB and
micromolar concentrations of Galb1-3GalNAc-OAllyl 17 (closed triangles, dotted line).
Figure 1. The eects measured for 1 and the ®rst
generation mimic 2 (Fig. 1(A)) con®rm the comparable
anity of the two ligands previously established by
ELISA inhibition assay.6 Furthermore, both substrates
appear to bind cooperatively to CTB, albeit with
dierent cooperativity factors. Indeed, binding of 1
to CTB is known to occur cooperatively.3e Using
calorimetric titrations, Freire and Schon3e have shown
that the intrinsic association constant of 1 for the B5
Acknowledgements
This work was supported by MURST and CNR.
References and Notes
1. Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed 1999, 38,
2300.
pentamer at 37 ꢀC is 1.05Â106 M , which increases by
1
2. (a) Spangler, B. D. Microbiol. Rev. 1992, 56, 622 and
references therein. (b) Masserini, M.; Freire, E.; Palestini, P.;
Calappi, E.; Tettamanti, G. Biochemistry 1992, 31, 2422.
3. (a) Lanne, B.; Schierbeck, B.; Angstrom, J. J. Biochem.
1999, 126, 226. (b) Lanne, B.; Schierbeck, B.; Karlsson, K. A.
J. Biochem. 1994, 116, 1269. (c) Angstrom, J.; Teneberg, S.;
Karlsson, K. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11859.
(d) Schengrund, C.-L.; Ringler, N. J. J. Biol. Chem. 1989, 264,
13233. (e) Schon, A.; Freire, E. Biochem. 1989, 28, 5019. (f)
Fukuta, S.; Magnani, J. L.; Twiddy, E. M.; Holmes, R. K.;
Ginsburg, V. Infect. Immun. 1988, 56, 1748.
4. (a) Merritt, E. A.; Sarfaty, S.; v.d. Akker, F.; L'Hoir, C.;
Martial, J. A.; Hol, W. G. J. Protein Sci. 1994, 3, 166. (b)
Merritt, E. A.; Sarfaty, S.; Jobling, M. G.; Chang, T.; Holmes,
R. K.; Hirst, T. R.; Hol, W. G. J. Protein Sci. 1997, 6, 1516.
(c) Merritt, E. A.; Hol, W. G. J. Curr. Opin. Struct. Biol. 1995,
5, 165, and references therein.
5. Bernardi, A.; Raimondi, L.; Zuccotto, F. J. Med. Chem.
1997, 40, 1855.
6. Bernardi, A.; Brocca, P.; Checchia, A.; Sonnino, S.; Zuc-
cotto, F. J. Am. Chem. Soc. 1999, 121, 2032.
7. Bernardi, A.; Boschin, G.; Checchia, A.; Lattanzio, M.;
Manzoni, L.; Potenza, D.; Scolastico, C. Eur. J. Org. Chem.
1999, 1311.
a factor of 4 if an adjacent binding site on CTB is
already occupied. In contrast, binding of 3±5 (Fig. 1(B))
does not display cooperative behavior. This suggests
that the communications between CTB monomers
elicited by 1 and 2 may be mediated by the NeuAc
residue, which is present in 1 and 2 and missing in 3±5.
In the series 3±5, the (R)-lactic acid derivative 4 (Figure
1(B), open triangles) displays the strongest anity for
CTB. The dissociation constants determined by non-
linear regression analysis are 667 mM for 3, 190 mM
for 4, and 1.1 mM for 5. For comparison, CTB has
a KD of about 40 mM and 81 mM for galactose and
lactose,12 respectively, and the titration curve of 2 mM
CTB with Galb1-3GalNAc-OAllyl 17 (Chart 1) is also
reported in Figure 1(B) (closed triangles). Asialo GM1,
devoid of the NeuAc moiety, showed no detectable
binding to CT at concentrations that were 320 times
higher than the IC50 of GM1.3d Thus the carboxy group
of 4, and to a lesser extent of 3, appears to have a
sizeable eect on the anity of the arti®cial receptors
for CT.
8. Bernardi, A.; Arosio, D.; Dellavecchia, D.; Micheli, F.
Tetrahedron: Asymmetry 1999, 10, 3403.
Further studies are in progress to determine the mode of
binding of 4, as well as to improve the anity by varying
the nature of the hydroxy acid side-chain. The hydroxy
acid-containing mimics reported in this paper are easily
accessible on a large scale and can be readily conjugated
to aglycons. Thus they may be used to build multivalent
ligands, which, ideally, should contain 5 pseudo-GM1
units capable of simultaneously interacting with the 5 B
units of the toxin.15
9. For a similar approach to the design of sialyl Lewis X mimics,
see: Kolb, H. C.; Ernst, B. Chem. Eur. J. 1997, 3, 1571.
10. Calculations were performed using MacroModel (Moha-
madi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Cau®eld, C.; Chang, G.; Hendrickson, T.; Still,
W. C. J. Comp. Chem. 1990, 11, 440) and following the pro-
tocols described for 1 and 2 in refs 5 and 6.
11. David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.