C O M M U N I C A T I O N S
Figure 5. Proposed mechanism for the catalysis.
dextrin itself. For this reason, nucleophilic catalysis is unlikely.
The role of the cyano groups must be to draw electrons away from
the OH groups, making them more acidic. This is supported by
the observation that 11 is the second best catalyst. In this compound,
the cyano groups have been replaced by OH groups, which are
electron-withdrawing though less so. Thus, an increased acidity of
these OH groups appears a crucial factor. This fits a role of the
OH groups acting as general acids. We therefore propose a
mechanism for the catalysis as outlined in Figure 5. A cyanohydrin
OH group donates a proton to the exocyclic oxygen facilitating
cleavage.
The cyclodextrin cyanohydrin 3 is an encouragingly potent
catalyst and appears to mimic part of the mechanistic apparatus of
natural glycosidases though with an entirely different functionality,
the cyanohydrin. Including other parts of this apparatus such as
nucleophilic catalysis is likely to improve the catalysis further.
Figure 4. Progress curve for the hydrolysis of 2 (10 mM) at pH 7.4, 59
°C in the presence of different concentration of 3 (0.01-0.1 mM).
were prepared by hydrogenolysis of the known stereoisomeric
benzyl-protected allyl derivatives,4 while dialdehyde hydrate 11 was
obtained by hydrogenolysis of 4. Dinitrile 12 was obtained by
conversion of the corresponding known benzyl-protected A,D diol8
into a diiodide with Ph3P, I2, and imidazole (91%) followed by
substitution with KCN (85%) and hydrogenolysis (100%, see
Supporting Information).
The catalytic power of these compounds toward 2 hydrolysis is
shown in Table 2. The propyl analogues 8, 9, and 10 afford no
catalysis, similar to â-cyclodextrin and regardless of whether the
two OH groups point toward or away from the cavity. This shows
that the cyano groups are essential. Dinitrile 12 is catalytic, but
with a 250 times lower catalytic power, showing that the two
cyanohydrin OH groups are very important for the catalysis. Finally,
the dialdehyde 11, which NMR shows is exclusively on dihydrate
form in aqueous solution, is a catalyst with a catalytic efficacy of
20 times lower than 3.
Acknowledgment. This work has been supported by the
Lundbeck Foundation, The Danish National Science Research
Council, and the Ramo´n Areces Foundation (F.O.C.).
Supporting Information Available: Synthetic procedures for
synthesis of 3 and 8-12, procedures for kinetic analysis, and Hanes
plots. This material is available free of charge via the Internet at http://
pubs.acs.org.
Table 1. Kinetic Parameters for the 3-Catalyzed Hydrolysis of
Various Glycosides in the Presence of 0.42 mM 3 at pH 7.4 and
59 °C
1
substrate
K
m (mM)
kcat
(
×
105 s-
)
kcat/kuncat
References
4-nitrophenyl-â-D-glucoside (2)
4-nitrophenyl-R-D-glucoside
4-nitrophenyl-R-D-mannoside
4-nitrophenyl-R-D-galactoside
2-nitrophenyl-â-D-galactoside
5.4
12
2.8
1.0
4.2
3.0
2.9
1.8
2.3
6.7
1047
2147
283
486
755
(1) (a) Dugas, H. Bioorganic Chemistry, 3rd ed.; Springer-Verlag: New York,
1996. (b) Wolfenden, R. Acc. Chem. Res. 2001, 34, 938-945.
(2) For research on enzyme mimics, see: (a) Breslow, R.; Dong, S. D. Chem.
ReV. 1998, 98, 1997-2011. (b) Kirby, A. J. Angew. Chem., Int. Ed. Engl.
1994, 33, 551-553. (c) Murakami, Y.; Kikuchi, J. I.; Hisaeda, Y.;
Hayashida, O. Chem. ReV. 1996, 96, 721-758. (d) Motherwell, W. B.;
Bingham, M. J.; Six, Y. Tetrahedron 2001, 57, 4663-4686. (e) Breslow,
R. Acc. Chem. Res. 1995, 28, 146-153. (f) Breslow, R.; Schmuck, C. J.
Am. Chem. Soc. 1996, 118, 6601-6605. (g) Breslow, R.; Zhang, B. J.
Am. Chem. Soc. 1992, 114, 5882-5883. (h) Akiike, T.; Nagano, Y.;
Yamamoto, Y.; Nakamura, A.; Ikeda, H.; Veno, A.; Toda, F. Chem. Lett.
1994, 1089-1092. (i) Breslow, R.; Zhang, B. J. Am. Chem. Soc. 1994,
116, 7893-7894. (j) Milovic, N. M.; Badjic, J. D.; Kostic, N. M. J. Am.
Chem. Soc. 2004, 126, 696-697. For previous work on glycosidase
mimics, see: (k) Doug, T. H.; Chou, J. Z.; Huang, X.; Bennet, A. J. J.
Chem. Soc., Perkin Trans. 2 2001, 83-89. (l) Ohe, T.; Kajiwara, Y.; Kida,
T.; Zhang, W.; Nakatsuji, Y.; Ikeda, I. Chem. Lett. 1999, 921-922.
(3) Rousseau, C.; Nielsen, N.; Bols, M. Tetrahedron Lett. 2004, 45, 8709-
8711.
On the basis of these results, the following can be elucidated
about the catalysis. Previous work on 6-C-substituted cyclodextrins
concluded that, on the basis of modeling and the highly variant
polarity of the 6S and 6R isomers, these derivatives have very
restricted conformational freedom along the C5-C6 bond as both
OH and alkyl substituents shun the tg conformation.4 Therefore,
an important feature in 3 is that the cyanohydrin 6-OH groups are
fixed in the gt conformation pointing toward the binding site. These
OHs are essential. However, their fixed conformation in itself is
not enough to promote catalysis as 9 is as uncatalytic as â-cyclo-
(4) Hardlei, T.; Bols, M. J. Chem. Soc., Perkin Trans 1 2002, 2880-2885.
(5) Angyal, S. J.; Le Fur, R. Carbohydr. Res. 1984, 126, 15-26.
(6) Normally an enzyme concentration that is significantly lower than substrate
concentration is assumed for the Michaelis-Menten equation to be valid.
However, the equation is also valid when KM ≈ Ks (Ks is the substrate
dissociation constant), which is the case here. Then d[ES]/dt ≈ 0 is also
fulfilled (steady-state conditions) over shorter time periods.
Table 2. Kinetic Parameters for the Catalysis by Different
Cyclodextrin Derivatives of the Hydrolysis of
4-Nitrophenyl-â-D-glucoside (2) at pH 7.4 and 59 °C
1
catalyst
K
m (mM)
kcat
(×
105 s-
)
kcat/kuncat
(7) Since kcat is comparatively small, KM is essentially identical to the
dissociation constant for the enzyme-substrate complex (Ks), and the
found value (15 mM) is close to the reported dissociation constant of 28
mM for this substrate to â-cyclodextrin.
3 (0.42 mM)
8-10 (2.1 mM)
11 (0.44 mM)
12 (2.2 mM)
5.4
-
7.6
6.3
3.0
-
0.14
1047
-
48
4
(8) Pearce, A. J.; Sinay¨, P. Angew. Chem., Int. Ed. 2000, 39, 3610-3612.
0.011
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