catalysis arises from the combined action of neutral and proto-
nated guanidine units, properly organized at the upper rim of a
cone calix[4]arene platform.
Acknowledgements
PRIN/MIUR 2008 and ATENEO La Sapienza 2010 are acknow-
ledged for financial support.
Fig. 3 Possible mechanisms for the catalytic cleavage of ATP pro-
moted by 1(H+)3: (a) nucleophilic catalysis; (b) general base catalysis.
GH+ = guanidinium.
Notes and references
1 (a) M. W. Hosseini, J.-M. Lehn and M. P. Mertes, Helv. Chim. Acta,
1983, 66, 2454–2466; (b) M. W. Hosseini, J.-M. Lehn, L. Maggiora,
K. B. Mertes and M. P. Mertes, J. Am. Chem. Soc., 1987, 109, 537;
(c) M. W. Hosseini, J.-M. Lehn, K. C. Jones, K. E. Plute, K. B. Mertes
and M. P. Mertes, J. Am. Chem. Soc., 1989, 111, 6330.
2 J. A. Aguilar, A. B. Descalzo, P. Díaz, V. Fusi, E. García-España,
S. V. Luis, M. Micheloni, J. A. Ramírez, P. Romani and C. Soriano,
J. Chem. Soc., Perkin Trans. 2, 2000, 1187 and references cited therein.
3 For an exhaustive review see: M. W. Hosseini, in Supramolecular Chem-
istry of Anions, ed. A. Bianchi, K. Bowman-James and E. Garcia-España,
Wiley VCH, New York, 1997, p. 421.
4 L. Baldini, R. Cacciapaglia, A. Casnati, L. Mandolini, R. Salvio,
F. Sansone and R. Ungaro, J. Org. Chem., 2012, 77, 3381.
5 M. Galli, J. A. Berrocal, S. Di Stefano, R. Cacciapaglia, L. Mandolini,
L. Baldini, A. Casnati and F. Ugozzoli, Org. Biomol. Chem., 2012, 10,
5109 and references cited therein.
6 (a) X. Fu and C.-H. Tan, Chem. Commun., 2011, 47, 8210;
(b) J. E. Taylor, S. D. Bull and J. M. J. Williams, Chem. Soc. Rev., 2012,
41, 2109.
7 D. O. Corona-Martinez, O. Taran and A. K. Yatsimirsky, Org. Biomol.
Chem., 2010, 8, 873.
8 R. Salvio, R. Cacciapaglia and L. Mandolini, J. Org. Chem., 2011, 76,
5438.
9 K. Ariga and E. V. Anslyn, J. Org. Chem., 1992, 57, 417.
10 In 80% DMSO the pKw for water autoprotolysis is 18.4 (see ref. 11). This
implies that 9.2 corresponds to neutral pH and, consequently, that pH 9.8
indicates a slightly basic solution, only 0.6 pH units above neutrality.
11 M. M. Kreevoy and E. H. Baughman, Phys. Chem., 1974, 78, 421–423.
12 Too much emphasis cannot be placed on exact figures, because distri-
bution diagrams were calculated on the basis of titration experiments
carried out at 25 °C, but no allowance was made for the temperature
change from 25 °C to 80 °C and, more importantly, for the perturbation
of equilibria caused by association of ATP with the various forms of the
catalyst.
trifunctional calix[4]arene in its diprotonated form 2(H+)2 (entry
4), whose catalytic efficiency is one order of magnitude lower
than that of 1 at pH 9.8. Not surprisingly, still lower is the
activity of the monoprotonated forms of regioisomeric bifunc-
tional calix[4]arenes 3H+ and 4H+ (entries 5 and 6, respectively),
where the electrostatic driving force for binding to ATP is
reduced to a minimum.
It is interesting to compare the catalytic efficiency of 1 with
that of [24]N6O2, although a strict comparison is not straight-
forward because of the different experimental conditions. From a
Michaelis–Menten analysis of the dephosphorylation of ATP to
ADP and P catalyzed by [24]N6O2 in a water solution (pH 7.0,
70 °C), Lehn and Mertes1c give a value of 0.064 min−1 for the
turnover frequency kcat,16 and one of 1 × 10−4 M for the Michaelis
constant KM. For the reaction catalyzed by 1 we find kcat
=
1.5 × 10−2 s−1 or 0.90 min−1, and KM = 1/K = 4 × 10−3 M.
Thus, while [24]N6O2 binds to ATP more strongly than 1, the
latter is a more efficient catalyst in terms of turnover frequency.
However, under subsaturating conditions, where reactivity is
determined by the kcat/KM ratio, a reversal in catalytic efficiency
is observed, with a modest superiority of [24]N6O2 over 1 (kcat
M = 640 M−1 min−1 for [24]N6O2 and 230 M−1 min−1 for 1).
In conclusion, the picture which emerges from the kinetic data
/
K
reported in this work is one in which the driving force for
optimal binding and activation is provided by the electrostatic
attraction between the triply charged anionic substrate and the
triply charged cationic catalyst 1(H+)3, possibly reinforced by
hydrogen bonding interactions. Consistent with this picture is the
marked decrease in catalytic efficiency accompanying a decrease
in the number of positive charges in the catalyst. Also consistent
with the data is the notion that the presence of a neutral guani-
dine is a necessary structural requisite for catalysis. Although no
direct evidence of the intermediacy of a phosphorylated catalyst
was obtained, by analogy with Lehn and Mertes’ conclusions,1 it
seems likely that the neutral guanidine unit acts as a nucleophilic
catalyst (Fig. 3a). The operation of turnover catalysis implies that
the phosphorylated intermediate, if any, is not stable under the
reaction conditions, but undergoes fast reaction with water to
give inorganic phosphate and free catalyst. Albeit less likely, a
mechanism in which the nucleophilic species is a water molecule
activated by a guanidine general base is equally consistent with
the kinetics and may be viewed as an alternative possibility
(Fig. 3b).
13 The common practice of adding a large excess of inert salt to keep the
ionic strength constant presents conceptual difficulties for solutions con-
taining multiply charged ions, because ion association may become
important at high salt concentration. For that reason only a moderate
amount of Me4NClO4 (0.1 M) was added. Neglecting the contributions
from the minute amounts of catalyst, we calculate I = 0.060 at the start of
the reaction and I∞ = 0.040 at the end of the reaction in the absence of
added salt. In its presence, the corresponding values are Io = 0.160 and
I∞ = 0.140. The amount of added salt is not large enough to keep the
ionic strength constant, but has the merit of keeping its variation within
narrow limits. For the same reason, the initial concentration of ATP was
kept constant in all kinetic runs, because changes in ATP concentration
would produce large changes in the ionic strength and thereby change the
rate in a way which has nothing to do with the order of the reaction.
14 In a control experiment, 10 mM ADP was exposed to the action of
0.2 mM 1 in 80% DMSO at 80 °C, pH 9.8, in the presence of 0.1
M Me4NClO4. The initial rate of ADP hydrolysis, vo = 5.6 × 10−8 M s−1
,
is 1/36 of the initial rate of ATP hydrolysis (vo = 2.0 × 10−6 M s−1) under
the same conditions.
15 For example, in the run in which Ccat = 0.4 mM (Fig. 2), the first half-life
period is 1300 s, but the second half-life period (from 50% to 75% reac-
tion) is 890 s.
16 Direct measurements of kcat under conditions where [24]N6O2 is fully
saturated with ATP gave a value of 0.060 min−1 at 80 °C, pH 7.0. Similar
values were obtained for other macrocyclic polyamines under the same
conditions (see ref. 1b).
To sum up, the catalytic efficiency of the triprotonated tetra-
guanidinocalix[4]arene 1(H+)3 in the cleavage of ATP rivals
those of [24]N6O2 and related macrocyclic polyamines. Effective
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 8941–8943 | 8943