1678 J. Am. Chem. Soc., Vol. 119, No. 7, 1997
Zhang and Breslow
Table 2. Hydrolysis of p-Nitrophenyl 3-Indolepropionate (31) at
37 °Ca
Scheme 5
c
d
entry
catalyst
M(II) pHb
kobs (s-1
)
krel
1
2
3
4
8.0 1.0 × 10-7
1
3
4
Cu(II) 8.0 3.2 × 10-7
Cu(II) 8.0 3.7 × 10-7
bipyridine
bipyridyl dimer 1 Cu(II) 8.0 (1.04 ( 0.02) × 10-3 10 400
5
6
7
8
9
7.0 3.0 × 10-8
Cu(II) 7.0 2.5 × 10-7
7.0 7.1 × 10-7
1
8
24
25
60
â-cyclodextrin
â-cyclodextrin
bipyridyl dimer 1
Cu(II) 7.0 (7.39 ( 0.09) × 10-7
7.0 (1.81 ( 0.42) × 10-6
10 bipyridyl dimer 1 Cu(II) 7.0 (5.49 ( 0.40) × 10-4 18 300
a Reactions were performed at 37 °C in 10 mM HEPES buffer
solution, in 1.0 × 10-4 M bipyridyl dimer 1, cyclodextrin, and
bipyridine; 5.0 × 10-5 M p-nitrophenyl 3-indolepropionate (31), 5.0
× 10-4 M CuCl2. b The pH was checked at the beginning and the end
of each run to hold to within (0.1 unit. c The rate constants were
obtained by analyzing the data using the Kore program and are averages
of at least two runs. All correlation coefficients were g0.9999. d Rate
constants relative to background (no catalyst).
Table 1. Binding Constants of Guests with Dimer 1 in Watera
Table 3. Hydrolysis of Esters Catalyzed by Bipyridyl
Cyclodextrin Dimer 1 in the Presence of Cu(II) at 37 °Ca
guest
Ka with Zn(II) (M-1
)
Ka (M-1
)
Ka(Zn(II))/Ka
carbonate 27 (7.70 ( 0.10) × 106 (1.43 ( 0.20) × 106
phosphate 28 (5.47 ( 0.17) × 107 (1.00 ( 0.34) × 106
phosphate 29 (1.02 ( 0.05) × 109 (2.05 ( 0.24) /x× 107
a Values determined by competition with DiTNS 30 (see refs 2-5)
in 10 mM HEPES buffer solution (pH 7.0) at ambient temperature (25
°C); average of more than three independent runs.
5
55
50
b
c
d
entry substrate
pH
kobs (s-1
)
kun (s-1
)
krel
1
2
3
4
5
6
7
ester 34 8.0 ( 0.1 9.61 × 10-5 1.42 × 10-5
ester 32 8.0 ( 0.1 1.35 × 10-4 1.50 × 10-7
ester 33 8.0 ( 0.1 1.74 × 10-4 1.00 × 10-7
ester 31 7.0 ( 0.1 5.49 × 10-4 3.00 × 10-8
ester 31 8.0 ( 0.1 1.04 × 10-3 1.00 × 10-7
7
900
1 740
18 300
10 400
ester 21 7.0 ( 0.1 6.76 × 10-3 3.00 × 10-8 225 000
These results suggest that the two hydrophobic ends of the
ditopic guests bind strongly into the two cyclodextrin cavities
of dimer 1, and that for phosphodiesters there is an extra
interaction of the phosphate anion with the metal cation. This
artificial metalloenzyme is thus much like a true enzyme, in
that binding with substrate in the transition state is tighter than
in the ground state. This is the first example of an artificial
metalloenzyme effectively binding a tetrahedral, negatively
charged phosphate transition state analogue for hydrolysis of
an ester, and indicates that the bipyridyl dimer 1 should have
catalytic power for ester hydrolysis with metal ions. However,
the difference of the binding constants for the carbonate 27 and
the transition state analogue 28 with the Zn(II) complex of dimer
1 greatly underestimates the magnitude of the rate accelerations
we observed.
Ester Hydrolysis. The first investigation of ester hydrolysis
employed p-nitrophenyl 3-indolepropionate (31) under various
conditions in 10 mM HEPES buffer (pH 7.0 or pH 8.0) at 37
°C; the results are listed in Table 2. Comparison of the catalyzed
and uncatalyzed (entry 5) reaction rates obtained at pH 7.0
indicated enhancements of 18 300 for dimer 1 with Cu(II) (entry
11) and 60 for dimer 1 without Cu(II) (entry 10). When the
reaction catalyzed by 1 and Cu(II) (entry 4) was conducted at
pH 8.0, the rate enhancement was 10 400. With simple
â-cyclodextrin, the rate enhancement observed for the hydrolysis
of substrate 31 was only 25 (entries 8 and 9) with and without
Cu(II) at pH 7.0. With 2,2′-bipyridine (entry 7), the hydrolysis
rate of ester 31 increased by a factor of only 12 with Cu(II).
The large rate acceleration by our enzyme mimic clearly
comes from the combination of the binding sites (two hydro-
phobic cavities) and the catalytic site (metal ion ligand). The
two hydrophobic cavities bind the substrate and juxtapose the
ester functional group and the catalytic metal ion of the artificial
metalloenzyme. The Cu(II) complex of dimer 1 is a successful
catalyst for the hydrolysis of this ester and others (Vide infra)
under physiological conditions of temperature and pH, in
contrast to many enzyme model studies that have employed
extreme conditions.
ester 21 8.0 ( 0.1 1.20 × 10-2 1.80 × 10-7
66 700
a All solutions were 1.0 × 10-4 M bipy-dimer 1, 2.0 × 10-4
M
CuCl2, and 6.0 × 10-5 M substrates. Reactions were conducted in 10
mM HEPES buffer solution for substrates 21, 31, and 34. Reactions
were carried out in 60% 10 mM HEPES buffer and 40% DMSO
solution for substrates 32 and 33. Rate constants were obtained by
averaging two or more kinetic runs. Standard deviations were less than
8% of the rate constants in the table. b Reactions were monitored to
>95% completion. The rate constants were obtained by analyzing the
data using the Kore program; the correlation coefficients were g0.9999.
c kun were obtained by calculating the initial rate constant without
d
1-Cu(II). krel ) kobs/kun.
We have synthesized some other esters (Scheme 5) to study
their hydrolysis catalyzed by bipyridyl dimer 1 with Cu(II); their
pseudo-first-order rate constants are shown in Table 3. The
hydrolysis of 34 showed only a 7-fold rate enhancement (entry
1); it can bind into only one cyclodextrin unit of dimer 1. We
expected that ester 21, containing an adamantane group, should
bind more strongly to 1 than 31. In fact, the hydrolysis rate of
ester 21 with 1 and CuCl2 at pH 7.0 and 37 °C was 225 000
times (entry 6) faster than the rate of uncatalyzed hydrolysis.
When an excess of substrate 21 was employed, at least 50
turnovers were seen in the ester hydrolysis catalyzed by dimer
1 and CuCl2.
Esters 32 and 33 contain a tert-butylphenyl group which
should bind strongly with dimer 1. However, the solubility of
substrates 32 and 33 is poor in aqueous solution, so the
hydrolyses of esters 32 and 33 were performed in 40% DMSO-
buffer solution. This solvent system decreases the binding
ability of these esters with bipyridine dimer 1 (Vide infra), and
decreases kobs
.
Ester 26 is long enough for binding into two cavities of dimer
1 (Scheme 6). Since the solubility of ester 26 in water is poor,
the hydrolytic experiments of ester 26 were performed in 70%
methanol-aqueous buffer or DMSO-aqueous buffer solvent
systems. The results are listed in Table A (Supporting Informa-
tion) and plotted in Figure A (Supporting Information). The
data show that the rate constant for the Cu(II) complex of dimer