Origin of rate-acceleration in ester hydrolysis with metalloprotease mimics

Article abstract of DOI:10.1016/S0968-0896(99)00310-7

Mimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to the primary side of β-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydrolysis of p-ni

Full text of DOI:10.1016/S0968-0896(99)00310-7

Bioorganic & Medicinal Chemistry 8 (2000) 647±652
Origin of Rate-Acceleration in Ester Hydrolysis with
Metalloprotease Mimics
Dong H. Kim* and Soo Suk Lee
Center for Biofunctional Molecules and Department of Chemistry, Pohang University of Science and Technology,
Pohang 790-784, South Korea
Received 23 August 1999; accepted 26 November 1999
AbstractÐMimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to
the primary side of b-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydro-
lysis of p-nitrophenyl acetate (PNPA) and enhance the rate of hydrolysis reaction by almost 300-fold. The e€ective molarities (EM)
of the mimics range from 0.2 to 1.9 M. Origin of the rate acceleration was examined: the reactivity of Zn(II) complexes of [12]aneN3
[12]aneN4, and [14]aneN4 for hydrolyzing PNPA increases with increase in basicity of the zinc bound hydroxides [Zn(II)±OH],
yielding a linear Bronsted plot. Free hydroxide ®ts well on this plot. A similar plot was obtained with the enzyme mimics. The
Bronsted relationships indicate that the Zn(II)±OH in the catalytic systems hydrolyzes the ester by direct nucleophilic attack on the
ester carbonyl of cyclodextrin-bound but not Zn(II)-coordinated PNPA. # 2000 Published by Elsevier Science Ltd. All rights reserved.
Introduction
Recently, Kimura et al. reported that Zn(II) forms a stable
and structurally well de®ned complex with [12]aneN3 and
a molecule of water.3 The pKa value of the zinc bound
water molecule was determined to be 7.3 (Fig. 2).4 This
water molecule is thus a strong nucleophile comparable to
the Zn(II) bound water molecule of carbonic anhydrase
and the complex was thought to be an excellent mimic of
the enzyme. Furthermore, Kimura et al. demonstrated
that the Zn(II)±[12]aneN3 complex accelerates the hydro-
lysis of not only p-nitrophenyl acetate (PNPA) but also
methyl acetate in neutral aqueous medium.4
Carboxypeptidase (CPA) is a much studied zinc pro-
1
tease which cleaves polypeptide substrates and esters.
The essential constituents of the active site of the enzyme
are a hydrophobic pocket for substrate recognition and
binding, the zinc ion that is coordinated to two imidazole
nitrogens of His-69 and His-196 and the carboxylate of
Glu-72, and the catalytically essential carboxylate of
Glu-270.1 A water molecule is coordinated to the zinc
ion as the fourth ligand. The scissile peptide carbonyl
group of the incoming substrate is known to coordinate
also to the zinc ion, which results in the activation of the
carbonyl carbon for a nucleophilic attack. Much has
been learned about CPA through kinetic and mechan-
istic studies, site-directed mutangenesis, and X-ray
crystallography, but details of the chemistry that occurs
in the catalytic process is still a matter of debate.
We thought that [12]aneN3 can also mimic the three
ligands that coordinate to the Zn(II) at the active site of
Christianson and Lipscomb proposed that the zinc
bound water molecule that is activated by the metal ion
and the carboxylate of Glu-270 serves as a nucleophile,
attacking at the carbonyl carbon of the scissile peptide
bond of substrate to form a tetrahedral transition state
that collapses to products (Fig. 1).2
Figure 1. Schematic representation of the general base mechanism
proposed for the enzymatic hydrolysis of peptide substrate by car-
boxypeptidase A.
*Corresponding author. Tel.: +82-562-279-2101; fax: +82-562-279-
5877; e-mail: dhkim@vision.postech.ac.kr
0968-0896/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00310-7

648
D. H. Kim, S. S. Lee / Bioorg. Med. Chem. 8 (2000) 647±652
CPA and thus may be useful for the construction of
0
modi®cation to react with [12]aneN3 at 100 ꢀC in DMF
solution. The crude product, thus obtained, was puri®ed
by CM±Sephadex chromatography followed by recrys-
tallization from water/methanol. The structure and the
CPA models. However, since CPA bears the P1 hydro-
phobic pocket for substrate recognition and binding as
discussed above, it was thought to be necessary for the
mimics of CPA to have a binding pocket that is struc-
turally complimentary to the substrate in addition to the
Zn(II) ligand. b-Cyclodextrin (b-CD) is a cyclic oligo-
saccharide having the overall shape of a truncated
cone.5 The cone is hydrophobic and forms an inclusion
complex with a phenyl ring in water. Thus, b-CD was
thought to be a viable mimic of the hydrophobic pocket
at the CPA active site. In this paper we describe com-
pounds 4, 5,6 and 6 that are constructed as mimics of
CPA by linking a macrocyclicpolyamine to the primary
side of b-CD followed by the addition of Zn(II), and
their evaluation as CPA models.
1
purity of the product were con®rmed by H NMR, 13C
NMR, mass spectroscopy and elemental analysis. In a
similar fashion, compounds 2 and 3 were synthesized.
Zinc complexes, 4, 5 and 6 were prepared in situ by
adding an equimolar amount of zinc chloride to the pH
7.0 Tris bu€er solution containing the respective mac-
rocyclicpolyamine, i.e. 1, 2 and 3.
The enzyme mimic (catalyst) is thought to bind the
substrate to form a complex that undergoes a chemical
reaction to yield products with regeneration of the cat-
alyst (Scheme 1). The breakdown of the complex to the
product occurs with the ®rst-order rate constant kcat.
The rate constant for the conversion of the substrate
into the product in the absence of the catalyst is repre-
sented by kun. The observed ®rst order rate constant
(kobs) and the dissociation constant (Km) of the complex
may be expressed by eqs (1) and (2), respectively. In
Results and Discussion
Compound 1 was synthesized by allowing 6-deoxy-O-
tosyl-b-CD prepared by the literature method7 with a

D. H. Kim, S. S. Lee / Bioorg. Med. Chem. 8 (2000) 647±652
649
these equations, [P], [C]0 and [S]0 represent the con-
centration of the product, enzyme mimic and substrate,
respectively.
erate the rate of hydrolysis of ester by (1) activating the
carbonyl group (mechanism A), (2) activating the leaving
group (mechanism B), or (3) providing a metal±hydrox-
ide nucleophile (mechanism C).8 A combination of these
activations is also possible for the hydrolysis of a given
ester. In order to distinguish between the possible
mechanisms that might operate for the present system,
we analyzed the reactivities of the Zn(II)±cyclopolya-
mine complexes for hydrolyzing PNPA. The second
order rate constant for the Zn(II)±hydroxide promoted
hydrolysis of PNPA increases with increase in the basi-
city of the Zn(II)±hydroxide to generate a Bronsted plot
with b=0.35 (Fig. 5) and we found that zinc-free hy-
droxide ®ts well on this plot. This observation suggests,
È
É
d‰PŠ=dt ˆkobs ‰SŠ ‡‰CÁSŠ ˆ kun‰SŠ ‡kcat‰CÁSŠ
ꢀ1†
ꢀ2†
0
0
È
É
Km ˆ ‰CŠ ‡‰SŠ0 =‰CÁSŠ
0
Since ‰CÁSŠ ˆ ‰CŠ ‰SŠ0=Km, eq (1) may be rewritten to give
0
eq (3) which may be further transformed to eq (4):
Á
Á
kobs ˆ Kmkun ‡ kcat‰CŠ = Km ‡ ‰CŠ
ꢀ3†
ꢀ4†
0
0
ꢀkobs kun†=‰CŠ ˆ kobs=Km ‡ kcat=Km
0
The kinetic studies were carried out at 25 ꢀC in pH 7.0
bu€ered solution using PNPA as substrate. The hydro-
lysis of the substrate generates p-nitrophenolate which
was monitored spectrophotometrically at 400 nm.
Under the conditions of a large excess of catalyst over
the substrate (‰SŠ ˆ 1:0  10 4 M), good pseudo-®rst-
0
order rate constants (kobs) were obtained. The satura-
tion kinetics were observed for all three enzyme mimics
as shown in Figure 3, satisfying the Michaelis±Menten
kinetics required as enzyme mimics. We plotted the kobs
values for PNPA hydrolysis at varied concentrations of
mimic 4 against ꢁkobs kun†=‰CPA mimicŠ according to
1
eq (4) and obtained kcat and Km values of 7.72Â10 4 s
Figure 3. Plots of kobs versus concentrations of enzyme mimic (4, 5
and 6) in the hydrolysis of PNPA.
and 3.32 mM, respectively (Fig. 4) and are collected in
Table 1 along with the kinetic parameters obtained with
the other two mimics. Highest rate increase was
observed with 4 which accelerates the hydrolysis of
PNPA by 291-fold. In principle, metal ions can accel-
Figure 4. Plots of (kobs±kun)/[CPA mimic]0 versus kobs for the hydro-
lysis of PNPA by 4, 5 and 6. In the plot the slope of the straight line
represents 1=Km and the y-intercept corresponds to kcat=Km, the
second order rate constant.
Table 1. Kinetic constants for the hydrolysis of PNPA at pH 7.0
(Tris bu€er) and 25 ꢀC in the presence of the enzyme mimic (catalyst)
Figure 2. pKa values of the water molecule bound to Zn(II)-macro-
cyclicpolyamide.3
Catalyst
kcat
(s
Km
(mM) (s
kcat=Km
kcat
kun
=
EM
(M)
1
1
1
a
)
M )
3
Zn(II)±[12]aneN3
Zn(II)±[12]aneN4
Zn(II)±[14]aneN4
4
5
6
3.6Â10
1.8Â10
3
3
3
3
3
0.75Â10
233Â10
171Â10
34.6Â10
4
4
4
7.72Â10
6.21Â10
1.28Â10
3.32
3.64
3.70
291
234
48
0.21
0.35
0.17
aThe value of kun for the hydrolysis of PNPA was reported to be
2.65Â10 6 s 1 (Breslow, R.; Nesnas, N. Tetrahedron Lett. 1999, 40, 3335).
Scheme 1.

650
D. H. Kim, S. S. Lee / Bioorg. Med. Chem. 8 (2000) 647±652
strongly, that the mechanism for the hydrolysis reactions
involves direct nucleophilic attack of the metal-hydroxide
on the uncoordinated ester (mechanism C). The pKa value
of the Zn(II) bound water molecules in 4, 5 and 6 were
determined to be lower compared with the pKa of the
water molecule bound to the corresponding Zn(II)±
cyclicpolyamines, i.e. Zn(II)±[12]aneN3, Zn(II)±[12]aneN4
and Zn(II)±[14]ane N4, respectively by about 0.05 unit.
Figure 5 also shows the Bronsted plot (b=0.42) con-
structed using the pKa values obtained with the enzyme
mimics. It can be seen that the Co(III)-cyclen attached b-
CD that was reported by Akkaya and Czarnik9 also ®ts
reasonably well on the Bronsted plot. Hence, we hasten to
propose that the direct nucleophilic attack of the Zn(II)-
hydroxide on cyclodextrin-bound but not the Zn(II)-
coordinated ester is also in operation for the enzyme
mimic promoted hydrolysis of PNPA. Contrary to
expectation, the carbonyl group of PNPA in the present
models does not appear to be activated by the Zn(II).
The present results are in accord with the mechanism
that the zinc bound water molecule at the active site of
CPA is suciently nucleophilic and thus attacks directly
on the scissile bond of the substrate.2,10,11 It is worth
noting that by being the substrate inserted in the hydro-
phobic pocket of the b-CD the rate of ester hydrolysis is
enhanced by about two orders of magnitude (Fig. 5).
The mechanistic pathway for the hydrolysis of PNPA
by the mimics at neutral pH may be represented by
Figure 6. Firstly, the phenyl ring of the substrate ®ts in
the hydrophobic cone of b-CD to form a complex. A
nucleophilic attack by the Zn(II)-hydroxide on the ester
carbonyl carbon would then ensue to generate a tetra-
hedral transition state which collapses to the products.
Since we failed to observe catalytic turnover with the
present mimics, it appears that the p-nitrophenolate that
is formed remains mostly in the cone of b-CD. Analo-
gous reaction paths are also envisioned for the PNPA
hydrolysis in the presence of 5 or 6.
Figure 5. Bronsted plots for the hydrolysis of PNPA by enzyme mimics
and Zn(II)±cyclicpolyamine complexes. The second order rate con-
stants are corrected for Zn(II)±OH concentration. From ref 9.
Jencks, W.P.; Gilchrist, M. J. Am. Chem. Soc. 1968, 90, 2622.
a
b
Figure 6. Schematic representation of the PNPA hydrolysis catalyzed by 4.

D. H. Kim, S. S. Lee / Bioorg. Med. Chem. 8 (2000) 647±652
651
It is well known that catalytic groups can provide much
greater rate-acceleration in intramolecular reactions
than in intermolecular reactions. The e€ective molarity
(EM) de®ned as the ratio of the rate constant for the
intramolecular reaction to that of the corresponding
intermolecular reaction is a parameter that re¯ects the
e€ectiveness of a catalytic system.12 E€ective molarities
in the range of 105±108 M are common for intramol-
cular nucleophilic catalytic reactions. We were inter-
ested in ®nding out what the e€ective molarities would
be of the enzyme mimics for hydrolyzing PNPA. The
EM values of 0.21, 0.35 and 0.17 M are found for 4, 5
and 6, respectively (Table 1). Although the e€ective
molarities are large, they are not as enormous as in a
carefully designed covalent system. This is not surpris-
ing in view of the loose association between the sub-
strate and the enzyme mimics and the ¯exible linker
between the metal complex and b-CD.
and a light yellowish syrup was obtained. Upon the
addition of acetone (300 mL) to the residue a colorless
solid precipitated which was collected by suction ®ltra-
tion and washed with acetone. Recrystallization of the
crude product from water yielded an analytically pure
sample (1.8g, 30% yield) as a white crystal: mp 168±170 ꢀC
1
(dec.); H NMR (DMSO-d6) d 2.43 (3H, s), 3.33±3.57
(42H, m), 4.32 (6H, s), 4.84 (7H, s), 5.67 (14H, s), 7.43 (2H,
d), 7.76 (2H, d); 13C NMR (DMSO-d6) d 21.80, 59.76,
71.90, 72.88, 81.36, 101.73, 127.35, 129.66, 132.47, 144.56.
Mono-6-deoxy-6-(1,5,9-triazacyclododecanyl)-ꢀ-cyclo-
dextrin (1). Dried b-CD-6-OTs, (0.65 g, 0.5 mmol) was
dissolved in DMF (20 mL) and 1,5,9-triazacyclodode-
cane (0.43 g, 2.5 mmol) was added to this solution. The
resulting mixture was heated at 100 ꢀC for 5 h under N2
atmosphere. After completion of the reaction, the mix-
ture was evaporated to dryness in vacuo at 40 ꢀC and
acetone (100 mL) was added to the residue. The solid
thus formed was collected by suction ®ltration and
washed with acetone. The residue was puri®ed by CM-25
Sephadex chromatography eluting with water followed by
0.1 M NH4OH to give an analytically pure sample (0.46 g,
Conclusion
We have synthesized mimics of zinc proteases by brid-
ging Zn(II) complexes of macrocyclicpolyamines to b-
CD. These enzyme mimics exhibit saturation kinetics in
hydrolysis of PNPA as enzymes do, and promote the
hydrolysis reaction by almost 300-fold. The e€ective
molarities of these systems range from 0.17 to 0.35 M.
From the analysis of the Bronsted plots obtained with
the mimics and the Zn(II)-macrocyclicpolyamines for
the PNPA hydrolysis, it was concluded that the Zn(II)-
bound water molecule in the mimics attacks on the ester
carbonyl of the PNPA that is CD-bound but not Zn(II)-
coordinated.
72% yield) as a white solid mp 232 ꢀC (dec.); H NMR
1
(D2O) d 2.08±2.18 (6H, m), 3.27±3.34 (12H, m), 3.43±4.20
(42H, m), 5.06 (7H, s); 13C NMR (D2O) d 27.48, 47.93,
59.76, 71.90, 72.88, 81.36, 101.73; Fab-MS (m/z) 1288
.
(M+1); anal. calcd for C51H89N3O34 4.5H2O: C, 44.73;
H, 7.21; N, 3.07. Found: C, 44.41; H, 6.98; N, 3.12.
Mono-6-deoxy-6-(1,4,7,10-tetraazacyclododecanyl)-ꢀ-cy-
clodextrin (2). This compound was synthesized in 64%
yield by the same procedure as described above mp
246 ꢀC (dec.); H NMR (D2O) d 2.73±2.78 (16H, m),
1
3.45±4.18 (42H, m), 5.07 (7H, s); 13C NMR (D2O) d
46.03, 59.66, 71.88, 73.08, 81.38, 101.79; Fab-MS (m/z)
.
1289 (M+1); anal. calcd. for C50H88N4O34 6.5H2O: C,
42.70; H, 7.24; N, 3.98. Found: C, 42.59; H, 7.06; N,
3.99.
Experimental
Melting points (MP) were determined on a Thomas±
Hoover capillary MP apparatus and are uncorrected.
1H NMR and 13C NMR spectra were recorded on a
Bruker FT-NMR spectrometer (300 or 500 MHz) and
chemical shifts are expressed in ppm relative to tetra-
methylsilane. Samples were dissolved in a mixture of
deuterium oxide and deuteriomethylsulfoxide. Low
resolution mass spectra were obtained with a KRATOS
MS 25 RFA spectrometer. Kinetic study was carried
out using a Perkin±Elmer HP 8453 UV±vis spectro-
meter. Elemental analyses were performed at POST-
ECH (CBM), Pohang, South Korea. Puri®cation of
cyclodextrin derivatives were performed by CM-25
Sephadex chromatography. All chemicals were of
reagent grade obtained from Aldrich Chemical Co.
N,N-Dimethylformamide was distilled over magnesium
sulfate and stored under nitrogen.
Mono-6-deoxy-6-(1,4,8,11-tetraazacyclotetradecayl)-ꢀ-
cyclodextrin (3). This compound was synthesized in
52% yield by the same procedure as described above:
mp 254 ꢀC (dec.); H NMR (D2O) d 1.78±1.84 (4H, m),
1
2.75±2.80 (8H, m), 2.83±2.90 (8H, m), 3.45±4.20 (42H,
m), 5.09 (7H, s); 13C NMR (D2O) d 29.85, 40.63, 42.82,
60.06, 70.98, 73.08, 81.86, 101.51; Fab-MS (m/z) 1317
(M+1); anal. calcd. for C52H92N4O34ꢂ 6H2O: C, 43.00;
H, 7.22; N, 3.86. Found : C, 42.87; H, 7.07; N, 3.92.
Kinetics
Hydrolysis rate of p-nitrophenyl acetate in aqueous
solution in the presence of an enzymic mimic was mea-
sured by following the increase in the 400 nm absorp-
tion using a computer-linked UV spectrometer. The
reaction solution was maintained at 25 ꢀC using a buf-
fered solution containing 0.05 M Tris bu€er (pH 7.0),
the ionic strength of which was adjusted to 0.10 with
sodium chloride. The typical procedure was as follows:
the enzyme mimic, zinc chloride (both ®nal concentra-
tions of 1.0±10.0 mM) and p-nitrophenyl acetate (the
®nal concentration of 0.1 mM) in 5% acetonitrile
Mono-6-deoxy-6-(p-toluenesulfonyl)-ꢀ-cyclodextrin (ꢀ-
CD - 6- OTs). A solution of p-toluenesulfonyl chloride
(1.5 g, 7.9 mmol) in anhydrous pyridine (10 mL) was
added with stirring to a solution of b-CD (5.0 g,
4.4 mmol, dried under vacuum at 100 ꢀC for 12 h) dis-
solved in anhydrous pyridine (50 mL). After 5 h at 4 ꢀC,
pyridine was removed under reduced pressure at 40 ꢀC

652
D. H. Kim, S. S. Lee / Bioorg. Med. Chem. 8 (2000) 647±652
solution were mixed and the absorption at 400 nm was
recorded as a function of time. The observed rate con-
stants (kobs) were obtained directly from the computer
interfaced UV spectrometer. All experiments were run in
duplicate and the tabulated data represent the average of
these experiments.
2. Christianson, D. W.; Lipscomb, W. N. Acc. Chem. Res.
1989, 22, 62.
3. Kimura, E.; Koike, T. Comments Inorg. Chem. 1991, 11,
285.
4. Kimura, E.; Shiota, T.; Koike, T.; Shiro, M.; Kodama, M.
J. Am. Chem. Soc. 1990, 112, 5805.
5. Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry;
Springer-Verlag: Berlin, 1978: pp 34±41.
6. This compound has been reported in the literature (Rosen-
thal, M. I.; Czarnik, A. W. J. Inclusion. Phenom. 1991, 10, 119.
7. Matsui, Y.; Okimoto, A. Bull. Chem. Soc. Jpn. 1978, 51,
3030.
8. Chin, J. Acc. Chem. Res. 1991, 24, 145.
9. Akkaya, E. U.; Czarnick, A. W. J. Am. Chem. Soc. 1988,
110, 8553.
Acknowledgements
This work was supported by the Pohang University of
Science and Technology and the Korea Science and
Engineering Foundation. The authors express their
thanks to Professor Jik Chin for the valuable help in the
preparation of this manuscript.
10. Breslow, R.; Wernick, D. L. J. Am. Chem. Soc. 1976, 98,
259.
11. Auld, D. S.; Galdes, A.; Geoghegan, K. F.; Holmquist, B.;
Martinelli, R. A.; Ballee, B. L. Proc. Natl Acad. Sci. USA
1984, 81, 5041.
References and Notes
1. Guiocho, F. A.; Lipscomb, W. N. Adv. Protein Chem. 1971,
25, 1.
12. Ables, R. H.; Frey, P. A.; Jencks, W. P. Biochemistry;
Jones and Bartlett Publisher: Boston, 1992, pp 135±137.