Highly efficient phosphate diester transesterification by a calix[4]arene-based dinuclear zinc(II) catalyst

Full text of DOI:10.1021/ja9638770

2948
J. Am. Chem. Soc. 1997, 119, 2948-2949
Highly Efficient Phosphate Diester
Transesterification by a Calix[4]arene-Based
Dinuclear Zinc(II) Catalyst
Peter Molenveld, Susan Kapsabelis,
Johan F. J. Engbersen,* and David N. Reinhoudt*
Laboratory of Supramolecular Chemistry and Technology
UniVersity of Twente, P.O. Box 217
7500 AE Enschede, The Netherlands
ReceiVed NoVember 8, 1996
Various enzymes,1 including P1 nuclease, DNA polymerase
I, phospholipase C, and alkaline phosphatase, use the synergic
action of two metal centers for the hydrolytic cleavage of
phosphate ester bonds. Several research groups have taken the
challenge to mimic this process with relatively simple model
compounds in which two metal centers have been linked by a
spacer group (i.e., Co(III) by Chin2,3 and Czarnik,4,5 Cu(II) by
Chin,6,7 Zn(II) by Breslow,8 Kimura,9 and Komiyama,10 or
lanthanide(III) by Schneider11). Although occasionally sub-
stantial rate accelerations for phosphate ester hydrolysis have
been observed, low substrate binding and lack of turnover are
general problems encountered with simple model systems.
Calix[4]arenes12-14 have been recognized for many years as very
suitable building blocks for the construction of multifunctional
enzyme models,12,15,16 because of the possibility of spatial
preorganization of catalytic groups and substrate binding sites.
However, although many examples of calix[4]arene-based
supramolecular receptors are known,13,14 the only example of
a calix[4]arene-based enzyme model17 has been reported by
Mandolini et al.,16 who showed that a calix[4]arene modified
at the lower rim with a crown ether Ba(II) complex exhibits
transacylase activity.
reported for nuclease mimics using this substrate. Comparison
of 1 with monofunctionalized calix[4]arene 2 and reference
pyridine complex 3 shows that the high catalytic activity of 1
can be attributed to a faVorable contribution of the calix[4]-
arene moiety in substrate binding and catalytic synergic action
of the two Zn(II) centers.
Scheme 1. Synthesis of a Calix[4]arene-Based Dimeric
Ligand (R ) CH2CH2OEt)a
In this paper we present calix[4]arene 1, functionalized with
two Zn(II) centers at the distal positions of the upper rim, as
the first example of a dinuclear complex which shows both
strong binding to a phosphate diester substrate and high catalytic
activity. The presence of 0.48 mM of 1 induces a 23000-fold
rate enhancement in the catalytic cyclization of the RNA model
substrate 2-(hydroxypropyl)-p-nitrophenyl phosphate (HPNP,18
pH 7, 25 °C). This is the largest catalytic rate acceleration
a Key: 33% MeNH2 in EtOH, H2, 10% Pd/C, 71%; (b) 2-(bromo-
methyl)-6-(hydroxymethyl)pyridine,20 K2CO3, CH3CN, 64%; (c) SOCl2,
CH2Cl2, 100%; (d) Me2NH‚HCl, K2CO3, CH3CN, 65%.
The calix[4]arene-based dimeric ligand 7 was prepared
stepwise from diformyltetrakis(ethoxyethyl)calix[4]arene 419 and
2-(bromomethyl)-6-(hydroxymethyl)pyridine20 according to
Scheme 1. The monomeric ligand was prepared analogously,
starting from monoformyltetrakis(ethoxyethyl)calix[4]arene,19
and the ligand of reference complex 3 was obtained by reaction
of bis(bromomethyl)pyridine20 with dimethylamine. The cor-
responding Zn(II) complexes (1-3) were generated in situ at
0.48 mM in acetonitrile/20 mM HEPES buffer 1:1 (v/v) at 25
°C, the reaction conditions for the catalysis experiments.21 The
association constants for Zn(II) complexation were determined
by UV spectrometry and are ca. 1 × 105 M-1, which implies
that under the reaction conditions 85% of the Zn(II) is bound
to the (aminomethyl)pyridine ligands.22
(1) For recent reviews, see: (a) Stra¨ter, N.; Lipscomb, W. N.; Klabunde,
T.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1996, 35, 2024. (b) Wilcox, D.
E. Chem. ReV. 1996, 96, 2435.
(2) Williams, N. H.; Chin, J. J. Chem. Soc., Chem. Commun. 1996, 131.
(3) Wahnon, D.; Lebuis, A.-M.; Chin, J. Angew. Chem., Int. Ed. Engl.
1995, 34, 2412.
(4) Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1993, 115, 12165.
(5) Chung, Y.; Akkaya, E. U.; Venkatachalam, T. K.; Czarnik, A. W.
Tetrahedron Lett. 1990, 31, 5413.
(6) Young, M. J.; Chin, J. J. Am. Chem. Soc. 1995, 117, 10577.
(7) Wall, M.; Hynes, R. C.; Chin, J. Angew. Chem., Int. Ed. Engl. 1993,
32, 1633.
(8) Chapman, W. H., Jr.; Breslow, R. J. Am. Chem. Soc. 1995, 117, 5462.
(9) Koike, T.; Inoue, M.; Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1996,
118, 3091.
(10) Yashiro, M.; Ishikubo, A.; Komiyama, M. J. Chem. Soc., Chem.
Commun. 1995, 1793.
(11) Ragunathan, K. G.; Schneider, H.-J. Angew. Chem., Int. Ed. Engl.
1996, 35, 1219.
(12) Gutsche, C. D. Calixarenes; The Royal Society of Chemistry:
Cambridge, England, 1989.
(19) Arduini, A.; Fanni, S.; Manfredi, G.; Pochini, A.; Ungaro, R.; Sicuri,
A. R.; Ugozzoli, F. J. Org. Chem. 1995, 60, 1448.
(20) Newcomb, M.; Timko, J. M.; Walba, D. M.; Cram, D. J. J. Am.
Chem. Soc. 1977, 99, 6392.
(21) In a typical kinetic experiment, the ligand 7 (20 µL, 50 mM) and
the metal perchlorate (40 µL, 50 mM) were added to 2 mL of acetonitrile/
20 mM HEPES 1:1 (v/v) at 25 °C. After a couple of minutes equilibration
time, HPNP18 (4 µL, 100 mM) was injected into the cuvette. The observed
first-order rate constant kobsd (s-1) was calculated with the extinction
coefficient of p-nitrophenolate at 400 nm by the initial slope method (<5%
conversion).
(22) All solutions remained clear during the time of the kinetic
experiments. In the absence of ligand, precipitation of polymeric Zn(II)
hydroxide took place.
(13) Bo¨hmer, V. Angew. Chem., Int. Ed. Engl. 1995, 34, 713.
(14) Takeshita, M.; Shinkai, S. Bull. Chem. Soc. Jpn. 1995, 68, 1088.
(15) Atwood, J. L.; Orr, G. W.; Robinson, K. D.; Hamada, F. Supramol.
Chem. 1993, 2, 309.
(16) Cacciapaglia, R.; Casnati, A.; Mandolini, L.; Ungaro, R. J. Am.
Chem. Soc. 1992, 114, 10956.
(17) p-Sulfonatocalix[6]arenes (ref 17a) and p-(carboxyethyl)calix[n]-
arenes (n ) 5-8, ref 17b) have shown to be catalytically active in the
acid-catalyzed hydration of 1-benzyl-1,4-dihydronicotinamide: (a) Shinkai,
S.; Mori, S.; Koreishi, H.; Tsubaki, T.; Manabe, O. J. Am. Chem. Soc. 1986,
108, 2409. (b) Gutsche, C. D.; Alam, I. Tetrahedron 1988, 44, 4689.
(18) Brown, D. M.; Usher, D. A. J. Chem. Soc. 1965, 6558.
S0002-7863(96)03877-2 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 12, 1997 2949
Figure 2. pH vs rate profile for transesterification of HPNP (0.19 mM)
catalyzed by 1 (0.48 mM) in acetonitrile/20 mM HEPES 1:1 (v/v) at
25 °C.
Figure 1. Dependency of the transesterification rate catalyzed by 1,
on HPNP concentration at pH 7.0 (b) and 7.4 (9) ([1] ) 0.48 mM,
acetonitrile/20 mM HEPES 1:1 (v/v), 25 °C).
The catalytic activities of the Zn(II) complexes toward
transesterification of HPNP were studied in the pH range of
6.8-8.2 of the aqueous buffer portion of the reaction mixture.21
In the absence of metallocatalysts, HPNP reacts extremely
slowly; the initial rate at pH 7.0 indicates a half-life for the
p-nitrophenol release of approximately 300 days (kobsd ) 2.7
× 10-8 s-1). However, the reaction rate increases dramatically
(23 000 times) upon the addition of 0.48 mM of 1 to the solution
(half-life 18 min, kobsd ) 6.3 × 10-4 s-1).23 In a separate
experiment using a 4-fold excess of HPNP, 1 shows turnover
without significant loss of activity during completion of the
reaction, which demonstrates that no product inhibition occurs.
The catalytic activity of the mononuclear calix[4]arene Zn(II)
complex 2 is 50 times lower than that of 1, emphasizing the
importance of synergism of the two Zn(II) centers in 1. The
fact that mononuclear calix[4]arene complex 2 is still 6 times
more active than reference complex 3 indicates that the calix-
[4]arene moiety contributes to the catalysis, probably by
enhancing the formation of the catalyst-substrate complex.
Assuming product formation by a two-step pathway Viz., (i)
molecule or a more tightly bound hydroxide ion. At higher
pH, the fraction of catalyst with Zn(II) bound hydroxide ions
is increased and consequently Kassn decreases. On the other
hand, the effective nucleophile concentration increases due to
deprotonation of Zn(II) bound water molecules (or substrate
hydroxyl group), and this increases kcat.28,29 The optimum rate
at pH 7.5 reveals the relatively low pKa of water coordinated
to Zn(II) in 1,30 which may be due to the close proximity of
the hydrophobic calix[4]arene moiety and the presence of 50%
acetonitrile in the reaction mixture. Coates et al. already showed
that the pKa of water coordinated to Zn(II) and Cu(II) is lowered
in a hydrophobic environment.31
Calix[4]arene 1 is not catalytically active in the hydrolysis
of diethyl p-nitrophenyl phosphate, ethyl p-nitrophenyl phos-
phate, or p-nitrophenyl phosphate, which shows that the
â-hydroxyl group of HPNP is essential in the catalytic process.
Therefore, the most likely mode of catalysis is a bifunctional
mechanism in which one of the Zn(II) centers serves as Lewis
acid activator of the phosphate group and the other is involved
in activation of the nucleophilic â-hydroxyl group giving rise
to relatively fast intramolecular reaction.8
rapid pre-equilibration of the catalyst-substrate complex (Kassn
)
followed by (ii) rate-determining conversion of the substrate
within the complex (kcat), saturation kinetics must be observed
when binding of the substrate to the catalyst is sufficiently high.
Measurement of the rate as a function of substrate concentration
(Figure 1) reveals that catalyst 1 has a very high affinity for
the substrate. At pH 7.0, already 80% of the substrate is bound
to catalyst 1 at equimolar concentrations (0.48 mM), and
saturation is readily reached at higher substrate concentration.
Fitting of the experimental data according to steady state pseudo-
first-order kinetics gave values for Kassn of 5.5 × 104 M-1 and
for kcat of 7.7 × 10-4 s-1. The unusually high binding constant
may be the result of the synergic action and the directional
preorganization of the two metallo binding sites24 on the calix-
[4]arene as well as the capacity to adjust the receptor site by
low-energy conformational changes of the calix[4]arene
moiety.19,25-27 As is shown in Figure 1, increase of the pH to
7.4 results in higher reaction rates and a higher substrate
concentration before saturation is reached (Kassn ) 1.7 × 104
M-1, kcat ) 10 × 10-4 s-1).
In conclusion, the nuclease mimic 1, possessing two Zn(II)
centers at the upper rim of a flexible calix[4]arene, exhibits fast
and strong binding of HPNP, followed by efficient intramo-
lecular transesterification, overall resulting in fast catalytic
turnover under very mild conditions.
Further analysis of the catalytic activity of 1 yields a bell-
shaped dependency of the pH (Figure 2). This behavior6,8 can
be explained by opposing pH effects on catalyst-substrate
complex formation (Kassn) and conversion of the substrate within
this complex (kcat). Binding of the substrate to the Zn(II) centers
requires the displacement of either a Zn(II) bound water
Acknowledgment. This investigation was supported by the Neth-
erlands Organization for Scientific Research (NWO) with financial aid
from the Netherlands Foundation for Chemical Research (SON).
JA9638770
(23) The catalytic activity of different calix[4]arene-based dinuclear metal
complexes was found to follow the order Zn2+ . Co2+ > Ni2+ ≈ Cu2+
.
(28) Itoh, T.; Fujii, Y.; Tada, T.; Yoshikawa, Y.; Hisada, H. Bull. Chem.
Soc. Jpn. 1996, 69, 1265.
(24) No saturation kinetics could be observed for mononuclear calix[4]-
arene complex 2 up to 6 equiv of HPNP (pH 7).
(25) Grootenhuis, P. D. J.; Kollman, P. A.; Groenen, L. C.; Reinhoudt,
D. N.; van Hummel, G. J.; Ugozzoli, F.; Andreetti, G. D. J. Am. Chem.
Soc. 1990, 112, 4165.
(26) Harada, T.; Rudzinsky, J. M.; Osawa, E.; Shinkai, S. Tetrahedron
1993, 49, 5941.
(27) Scheerder, J.; Vreekamp, R. H.; Engbersen, J. F. J.; Verboom, W.;
van Duynhoven, J. P. M.; Reinhoudt, D. N. J. Org. Chem. 1996, 61, 3476.
(29) Kimura, E. Progress in Inorganic Chemistry; Karlin, K. D., Ed.;
John Wiley & Sons: New York, 1994; Vol. 41, p 443.
(30) Potentiometric titrations with complex 3 in acetonitrile/0.1 mM
aqueous NaClO4 1:1 (v/v) shows that complex formation, at pH 6-8, is
simultaneously followed by the titration of an additional proton in the pH
region of 7.5-8.5.
(31) Coates, J. H.; Gentle, G. J.; Lincoln, S. F. Nature (London) 1974,
249, 773.