Catalytic hydrolysis of p-nitrophenyl picolinate by copper(II) and zinc(II) complexes of N-(2-deoxy-β-D-glucopyranosyl-2-salicylaldimino)

Article abstract of DOI:10.1002/kin.10049

D-glucosamine Schiff base N-(2-deoxy-β-D-glucopyranosyl-2-salicylaldimino) and its Cu(II) and Zn(II) complexes were synthesized and characterized. The hydrolysis of p-nitrophenyl picolinate (PNPP) catalyzed by ligand and complexes was investigated kinetic

Full text of DOI:10.1002/kin.10049

Catalytic Hydrolysis
of p-Nitrophenyl Picolinate
by Copper(II) and Zinc(II)
Complexes of N-(2-Deoxy-
β-D-glucopyranosyl-2-
salicylaldimino)
XIANG YAN,2 XIANCHENG ZENG,1 SIQING CHENG,1 YANTUAN LI,1 JIAQING XIE1
1Department of Chemistry, Sichuan University, Chengdu 610064, People’s Republic of China
2Department of Environment Engineering, Beijing University of Aeronautics & Astronautics, Beijing 100083,
People’s Republic of China
Received 10 July 2000; accepted 14 December 2001
DOI 10.1002/kin.10049
ABSTRACT: D-glucosamine Schiff base N-(2-deoxy- -D-glucopyranosyl-2-salicylaldimino) and
its Cu(II) and Zn(II) complexes were synthesized and characterized. The hydrolysis of
p-nitrophenyl picolinate (PNPP) catalyzed by ligand and complexes was investigated kineti-
cally by observing the rates of the release of p-nitrophenol in the aqueous buffers at 25 C and
different pHs. The scheme for reaction acting mode involving a ternary complex composed of
ligand, metal ion, and substrate was established and the reaction mechanisms were discussed
by metal–hydroxyl and Lewis acid mechanisms. The experimental results indicated that the
complexes, especially the Cu(II) complex, efficiently catalyzed the hydrolysis of PNPP. The cat-
alytic reactivity of the Zn(II) complex was much smaller than the Cu(II) complex. The rate con-
1
stant kN showing the catalytic reactivity of the Cu(II) complex was determined to be 0.299 s
(at pH 8.02) in the buffer. The pKa of hydroxyl group of the ternary complex was determined to
C
be 7.86 for the Cu(II) complex. 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 345–350, 2002
recent years as model reactions of metalloenzymes
[1–4] such as carboxypeptidase A [5], carbonic anhy-
drase [6], and related enzymes. It is now well known
that the roles of a metal ion are to activate the substrate
as an electrophilic catalyst, stabilizing the negative
charges that are formed during the reactions, and to act
as a source of hydroxide ion at neutral or mild base
buffer [7–10]. In related metalloenzymes, the metal
ion appears to activate the seine hydroxyl group [11].
Thus it is important for model studies to examine the
INTRODUCTION
Metal-ion-complex-catalyzed reactions of carboxylic
acid derivatives have been extensively investigated in
Correspondence to: Zeng Xiancheng; e-mail: zengxc@pridns.
scu.edu.cn.
Contract grant sponsor: National Natural Science Foundation of
China.
Contract grant number: 29783031.
2002 Wiley Periodicals, Inc.
c

346
YAN ET AL.
cooperation between hydroxyl groups and metal ion in
catalysis.
From the above scheme, it can be expressed that
Most models studied so far involve a ligand and
one or two hydroxyl groups through complexation with
metal ion for catalytic hydrolysis of the title compound
[7,8]. Few examples report the model involving the lig-
and and multihydroxyl groups (such as sugar) through
complexation with metal ions, which can provide more
nucleophiles for attacking the substrate. On the other
hand, it had been reported, with a few examples, that
the complexes of sugar derivants with metal ions are
very important in developing new metal-affinity chro-
matographic material and chiral homogeneous cata-
lysts [12]. In this paper, the effect of the complexes
of D-glucosamine with Cu(II) and Zn(II) ions on the
hydrolysis of p-nitrophenyl picolinate (PNPP) are in-
vestigated, the scheme for acting mode established, and
the reaction mechanism discussed. The results indicate
that the complexes greatly catalyzed the hydrolysis of
PNPP.
Rate = k [S]T = kN[MLnS] + k0([S]T [MLnS])
␺
(4)
where k is the apparent rate constant for hydrolysis
␺
of substrate.
So from Eq. (2), we obtain
[MLnS] = KS[MLn]T[S]T/(1 + KS[MLn]T) (5)
Inserting Eq. 5 into Eq. 4 and rearranging, we obtain
1
1
1
1
=
+
(kN k0)KS [MLn]T
(6)
k
k0
kN k0
␺
From Eq. (6), it can be seen that the slope and the
1
1
intercept of the plot
KS values.
vs.
T afford the kN and
k
k0
[MLn]
␺
Suppose the acid dissociation equilibrium takes
place in the ternary complex in the buffers to
CuSG 2H2O.
MATHEMATICAL MODEL INVOLVING
METAL-ION-COMPLEX-CATALYZED
REACTION
For enzyme catalytic mechanisms, a ternary complex
(composed of ligand, metal ion, and substrate) which
has particularly low energy during the catalytic reac-
tion is suggested. The process of metal-ion-complex-
catalyzed hydrolysis of PNPP is described as follows:
A metal complex [MLn] forms a ternary complex
[MLnS] with a substrate [S] with an association con-
stant KS, and then an intracomplex nucleophilic sub-
stitution reaction takes place in the ternary complex
in the rate-limiting step, with a first-order rate con-
stant kN to afford the products P. The products are also
produced through spontaneous hydrolysis, without in-
volving a ternary complex [13]. The process can be
written as
where kNmax is the first-order rate constant for product
because of the discomposing of ternary complex, and
Ka is the acid dissociate constant of ternary complex.
If the dissociation equilibrium is reached rapidly,
then the reaction rate is determined by the amount of
dissociated complex anion so that we have
Ka
kN =
kNmax
(7)
[H]+ + Ka
Rearranging Eq. (7), we obtain
1
1
1
=
+
kNmax Ka
[H+]
(8)
kN
kNmax
According to Eq. (8), the kNmax and ka values can be
obtained from a plot of 1/kN vs. [H+].
Ks
KN
→
⇔
MLn + S MLnS
P
(1)
[MLnS]
KS =
[MLn][S]
EXPERIMENTAL SECTION
General Methods
[MLnS]
=
(2)
(3)
([MLn]T [MLnS])([S]T [MLnS])
Infrared spectra were obtained on a PE 983 IFS
FIOIR spectrophotometer. All elementary analyses
were performed on an EA1110 elementary analyzer.
Kinetic studies were carried out by UV–vis methods,
with a GBC 916 UV–vis spectrophotometer equipped
with a thermostatic cell holder. The contents of metal
k0
→
S
P
where k0 is the first-order rate constant for hydrolysis
of PNPP in buffer. [S]T and [MLn]T are the total con-
centrations of the substrate and complex respectively.

CATALYTIC HYDROLYSIS OF p-NITROPHENYL PICOLINATE
347
ion in complexes were demarcated by EDTA standard
solution.
Anal. Calcd for CuC13H19O8N: C, 40.83; H, 4.97;
O, 33.51; N, 3.66; Cu, 17.01; Found: C, 41.58; H,
4.85; O, 33.58; N, 3.53; Cu, 16.45. IR: OH, 3423
1
1
1
cm
;
N, 1628 cm
;
N, 372 cm
;
,
O
CH
1
Cu
Cu
Materials
225 cm
.
All reagents, unless otherwise indicated, were of an-
alytical grade and were used without further purifica-
tion. Glucosamine hydrochloride was of biochemical
grade and was used without further purification. Sali-
cylaldehyde was purified by distillation. p-Nitrophenyl
picolinate (PNPP) was supplied by the Organic Chemi-
cal Laboratory of Sichuan University [14]. PNPP stock
solution for kinetics was prepared in acetonitrile.
The water used for syntheses and kinetics was ob-
tained by distilling deionized water twice. To avoid
the influence of the chemical component of different
buffers, Tris–TrisH+ buffer was used in all cases and
its pH was adjusted by adding analytical pure nitron
acid in all runs.
ZnSG H2O. A solution of Zn(Ac)2 (10 mmol) in 20
ml methanol was added dropwise to a solution of
ligand (10 mmol) in 20 ml of methanol at 45 C. After
2 h of vigorous stirring, the solution was reduced to
10 ml through distillation. The light-yellow precipitate
that appeared after the addition of 20 ml of ether was
filtered and washed with ether. The complex was dried
overnight in vacuum.
Anal. Calcd for ZnC26H34O13N2: C, 48.22; H, 5.25;
O, 32.15; N, 4.33; Zn, 10.05. Found: C, 48.10; H, 5.35;
1
O, 32.46;N, 4.26;Zn, 9.83. IR: OH, 3352cm
;
,
N
CH
1
1
1
1619 cm
;
N, 345 cm
;
O, 226 cm .
Zn
Zn
The structures of ligand and the Cu(II) and Zn(II)
complexes are shown in Fig. 1. The ligand and the
complexes were all water-soluble in the buffer.
N-(2-Deoxy-␤-D-glucopyranosyl-2-
salicylaldimino)
(SG H2O). Glucosamine hydrochloride (10 mmol)
and sodium bicarbonate (12 mmol) were dissolved in
10 ml of water, and salicylaldehyde (10 ml) was added.
The mixture was stirred vigorously at room temper-
ature and in 10 min the separation of crystals com-
menced. After 3 h of stirring, the bright precipitate
was filtered, washed with cold water, and dried in vac-
uum [15]. The compound was found to melt sharply at
183 C.
Kinetics Studies
Solutions were prepared in the buffer. Reaction
temperature was maintained at 25 C. Release of
P-nitrophenol was monitored at 400 nm. Each ki-
netic run was initiated by injecting 15 ␮l of PNPP
3
3
(10 mol dm in CH3CN) into the cuvette contain-
ing 3 ml of the buffer solution. Rate constants were ob-
tained from the (linear) plots of log(A At) vs. time.
Each pseudo-first-order rate constant is the mean value
of five determinations; its average relative standard de-
viation is smaller than 1.5%.
Anal. Calcd for C13H17O6N: C, 55.12; H, 6.01; O,
33.92; N, 4.94; Found: C, 54.94; H, 6.18; O, 34.01; N,
4.86.
CuSG 2H2O. A solution of Cu(Ac)2 (10 mmol) in
20 ml methanol was added dropwise to a solution of
ligand (10 mmol) in 20 ml of methanol at 45 C. After
2 h of vigorous stirring, the solution was reduced to
10 ml through distillation. The grayish-green precipi-
tate that appeared after the addition of 20 ml of ether
was filtered and washed with ether. The complex was
dried overnight in vacuum [16].
RESULTS AND DISCUSSION
The Apparent Rate Constants of the
Hydrolysis of PNPP at pH 7.21 and 25 C
The ligand and the complexes are water-soluble and
so the kinetics were carried out in plain buffer for
all reagents. Table I shows the apparent rate constants
Figure 1 The structures of ligand and Cu(II) and Zn(II) complexes.

348
YAN ET AL.
Table I Apparent Rate Constants of the Hydrolysis
Table II pH Dependencies of kN and K S in Buffer at
of PNPP at pH 7.21 and 25 C
25 C for CuSG 2H2O
3
1
3
1
1
KS (103 mol dm3)
No.
System
k (10
s
)
␺
pH
kN (10
s
)
1
2
3
4
5
6
None
Cu2+
Zn
0.0575
5.566
0.383
0.944
19.75
1.271
7.21
7.60
7.75
7.90
8.02
43.96
58.89
87.69
105.8
298.6
8.142
1.474
0.985
0.598
0.321
SG H2O
CuSG H2O
ZnSG H2O
All data were measured in 10 2 mol dm Tris–TrisH+ (I = 0.2,
3
All data were measured in 10 2 mol dm Tris–TrisH+(I = 0.2,
3
5
3
KCl) buffer. [PNPP] = 5 10 mol dm
.
KCl) buffer at pH 7.21 and 25 C. The concentrations of all reagents
are 10 4 mol dm 3. [PNPP] = 5 10 5 mol dm
3
.
it can be seen that the ternary complex mathemati-
cal model proposed is reasonable, and the value of
kN and KS could be obtained from those straight
lines.
(k ) obtained under the condition of an excess reagent
␺
over the substrate at pH 7.21 and 25 C. The table indi-
cates that the ligand and complexes were reactive in the
hydrolysis of PNPP. The rate enhancement by ligand
alone is large, which is more than 18-fold of k0. It is
one of the phenomena in works of complexes imitating
hydrolytic metalloenzymes. The reactivity of the lig-
and to PNPP is probably relative to the space structure
of the ligand. The hydroxyls on the glucosamine act as
nucleophiles, attacking the substrate. So, though with-
out metal ions, the ligand still can catalyze the hy-
drolysis of PNPP. The large rate enhancement of
more than 400-fold is obtained by the addition of
10 4 mol dm 3 CuSG 2H2O. Even a more remarkable
rate enhancement was achieved with Cu(II) or Zn(II)
alone, but the catalytic abilities of complexes were
much more higher than metal ions alone. This sug-
gests that the formation of a ternary complex accel-
erated the leaving of p-nitrophenol in the aqueous
buffer.
Tables II and III list the rate constants and disso-
ciation constants, respectively, for complexes at 25 C
and different pHs. CuSG 2H2O is the most active com-
1
plex in reagents and the kN of it is to be 0.299 s at
25 C and pH 8.02, which is more than 5200-fold of k0.
With the different pH values, the plot of 1/kNvs. [H+]
is shown in Fig. 3.
According to Eq. (8), the pKa for the ternary com-
plex to CuSG 2H2O was obtained to be 7.86. The
pKa decreases from 14 to 7.86, meaning that the
metal-bound water molecule ionizes and yields a high
fraction of hydroxide at mild base buffer. Such a
metal-bound hydroxide ion is a potent nucleophile re-
taining most of the reactivity of free hydroxide ions.
The observed stoichiometry was 1:1 for CuSG 2H2O
and the proposed reaction mechanism is shown in
Fig. 4. In a ternary complex, the complex of PNPP
with Cu2+ involving pyridine nitrogen is very impor-
tant at the rate-limiting step of attacking a ligand-
oxide anion nucleophile on the substrate carbonyl
carbon to form an additional intermediate [17,18].
The intermediate was then disclosed, the product
was released, and the reaction restarted. In principle,
Determination of kN, KS, and Ka Values
As shown in Fig. 2, the plots of 1/(k
k0) vs.
␺
1/[MLn]T is a straight line. From those straight lines,
Figure 2 Plots of CuSG 2H2O 1/(k
k0) vs. 1/[MLn]T
␺
3
for the hydrolysis of PNPP in 0.01 mol dm Tris at 25 C
and at different values of pH: , 7.21; , 8.02; , 7.60,
respectively.
Figure 3 Plot of 1/kN vs. [H+] for the reaction of PNPP
with CuSG 2H2O in 0.01 M Tris.

CATALYTIC HYDROLYSIS OF p-NITROPHENYL PICOLINATE
349
3
Figure 4 Proposed mechanism of CuSG 2H2O catalysis of hydrolysis of PNPP in 0.01 mol dm Tris.
CuSG 2H2O could catalyze the hydrolysis of PNPP
by the metal hydroxide mechanism [10]. The impor-
tant roles of Cu(II) ion in the buffer is to activate
the substrate and produce hydroxide ions acting as
nucleophiles.
cyclodextrins [19–22]. Because hydroxide is hy-
drophilic, the ligand and complexes are easily soluble
in water. In aqueous solution, the complex depended on
hydrogen bonds and van der Waals forces between sub-
strateandcatalysttoholdthesubstratemoleculetightly,
and then the nucleophile attacked the carbonyl carbon
of PNPP. In some means, this behavior of complexes
imitates integration position of hydrolytic metalloen-
zyme. This is very important in designing artificial en-
zymes. The function of Zn(II) in the intermediate is to
stabilize the structure of ternary complex and stabilize
the negative charges.
In summary, the Cu(II) and Zn(II) complexes of N-
(2-deoxy-␤-D-glucopyranosyl-2-salicylaldimino) are
good catalysts for hydrolysis of PNPP in the plain
buffer at 25 C. Although Cu2+ and Zn2+ had cat-
alytic reactivity to the hydrolysis of PNPP, their com-
plexes had higher activity than metal ions alone, and
the catalytic ability of CuSG 2H2O is much larger than
that of ZnSG H2O. So multihydroxyl metal complexes
have extensive prospects in the field of artificial hy-
drolytic metalloenzymes.
As we know, hydrolytic metalloenzymes in nature
generally bind their substrates and then use the ac-
tion of two or more well-placed functional groups to
achieve catalysis. The observed stoichiometry is 1:2 for
ZnSG H2O. In Fig. 1, it is shown that Zn(II) complexes
have no empty space for coordinating with PNPP, and
it’s well known that the ability of Zn(II) to activate
water molecules to be nucleophile is much smaller
than Cu(II) [18], and so the activity of ZnSG H2O
was smaller than that of CuSG 2H2O. But the kN of
ZnSG H2O is 85-fold of the rate constant of the PNPP
divided spontaneously at pH 8.02 and 25 C in the
buffer. The important reason why ZnSG H2O could
catalyze the hydrolysis of PNPP probably is the hy-
droxides of glucosamine acting as nucleophiles to at-
tack carbonyl carbon of substrate to form an acylation
intermediate, as shown in Fig. 5. Then the acylation
intermediate released acyl, and the reaction was fin-
ished and restarted. So, the kN of ZnSG H2O also can
be looked as the constant of deacylation. This pro-
posed catalytic mechanism was similar to those of
Table III pH Dependencies of kN and KS in Buffer at
25 C for ZnSG H2O
3
1
1
pH
k (10
s
)
KS (103 mol dm3)
7.10
7.21
7.60
8.02
1.155
3.145
17.63
36.74
3.851
2.374
2.403
2.613
All data were measured in 10 2 mol dm 3 Tris–TrisH+ (I = 0.2,
Figure 5 Proposed mechanism of ZnSG H2O catalysis of
hydrolysis of PNPP in 0.01 mol dm Tris.
3
5
3
KCl) buffer. [PNPP] = 5 10 mol dm
.

350
YAN ET AL.
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