Syntheses of a linear pentadentate ligand and its zinc(II) complex and its promoted hydrolysis of 4-nitrophenyl acetate

Full text of DOI:10.1081/SIM-100002039

Synthesis and Reactivity in Inorganic and Metal-Organic
Chemistry
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SYNTHESES OF A LINEAR PENTADENTATE LIGAND
AND ITS ZINC(II) COMPLEX AND ITS PROMOTED
HYDROLYSIS OF 4-NITROPHENYL ACETATE
Yongshu Xie , Qingliang Liu , Xiaolong Xu , Jun Yu & Ruisen Lin
To cite this article: Yongshu Xie , Qingliang Liu , Xiaolong Xu , Jun Yu & Ruisen Lin (2001)
SYNTHESES OF A LINEAR PENTADENTATE LIGAND AND ITS ZINC(II) COMPLEX AND
ITS PROMOTED HYDROLYSIS OF 4-NITROPHENYL ACETATE, Synthesis and Reactivity in
Inorganic and Metal-Organic Chemistry, 31:2, 195-204, DOI: 10.1081/SIM-100002039
To link to this article: http://dx.doi.org/10.1081/SIM-100002039
Published online: 15 Feb 2007.
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SYNTH. REACT. INORG. MET.-ORG. CHEM., 31(2), 195–204 (2001)
SYNTHESES OF A LINEAR
PENTADENTATE LIGAND AND ITS
ZINC(II) COMPLEX AND ITS PROMOTED
HYDROLYSIS OF 4-NITROPHENYL
ACETATE
Yongshu Xie,1 Qingliang Liu,1,∗ Xiaolong Xu,1 Jun Yu,1
and Ruisen Lin2,∗
1Department of Chemistry, University of Science and
Technology of China, Hefei, Anhui 230026, P. R. China
2Department of Chemistry, Zhejiang University, Hangzhou,
Zhejiang 310027, P. R. China
ABSTRACT
A linear pentadentate ligand, N-(2-hydroxyethyl)-Nꢀꢀ-(2-hydroxy-
benzyl)-diethylenetriamine (HL), and its zinc(II) complex were
synthesized and characterized by IR spectra, 1H NMR spectra and
elemental analyses. By pH potentiometric titration, protonation
constants of HL and stability constants of Zn(II) complexation
with HL have been determined at 25 0.1◦C and I = 0.1 mol/L
(KNO3). The kinetics of 4-nitrophenyl acetate (NA) hydrolysis
catalyzed by the complex was determined spectrophotometrically
at 25 0.1◦C and I = 0.1 mol/L (KNO3) in CH3CN (10 % v/v)
at pH 7.0–9.04 (50 mmol/L buffers), and the second-order rate
constants kc for NA hydrolysis were obtained. The experimental
∗Address correspondence to Qingliang Liu (E-mail: qliu@us) or Ruisen Lin.
195
Copyright ꢁ 2001 by Marcel Dekker, Inc.
www.dekker.com
C

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XIE ET AL.
results indicate that Zn···−OR (R = alkyl) is a better nucleophile
than Zn···−OAr (Ar = aryl). A good catalysis was realized with a
kc value of 8.5 × 10−2 mol−1 · L · s−1 at pH 9.04.
INTRODUCTION
Hydrolytic enzymes often use external H2O or internal alcoholic residues
(e.g. serine)asnucleophilestoreactwithelectrophilicsubstrates(e.g. carboxyesters
and phosphates), wherein the prior activation of the nucleophiles and/or elec-
trophiles of the substrates is essential (1). For instance, in alkaline phosphatase
(AP) (2), the Zn(II)-bound alcoholic OH (Ser102) deprotonates to yield the good
nucleophile Zn···−OR which initially attacks the phosphate substrates.
The model studies (3,4) of Zn(II) complexes have indicated that Zn···−OAr
can also act as nucleophiles to catalyze the hydrolysis of 4-nitrophenyl acetate.
The model studies mainly focus on macrocyclic (5–7) and tripodal (8,9) lig-
ands, but there are only a few works dealing with linear ligands (10–13). Herein
we report the synthesis of a linear N-(2-hydroxyethyl)-Nꢀꢀ-(2-hydroxybenzyl)-
diethylenetriamine (HL) as shown in Figure 1. Its protonation and complexation
equilibria with Zn(II) were studied by pH titration. Thus, a distribution diagram of
the system at various pH values was obtained. The kinetics of the 4-nitrophenyl ac-
etate hydrolysis catalyzed by the complex was investigated spectrophometrically.
Values of the rate constants (kc) of the catalyzed hydrolysis reaction were ob-
tained. Since HL contains both phenoxy and alkoxy groups, different nucleophiles,
Zn···−OAr and Zn···−OR, may be yielded upon coordination with a Zn(II) ion. By
comparing the kc values with the distribution diagram, the relative nucleophility
and the catalytic capacity for the various nucleophiles may be found. Based on
these results, the mechanism of the hydrolysis reaction is discussed.
H
N
NH
HN
NH
OH
HN
3HCl
3HCl
HO
NH2 HO
1
(L
3HCl
3HCl)
(HL
)
Figure 1. The structures of L1 · 3HCl and HL · 3HCl.

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197
EXPERIMENTAL
Materials, Instruments, and pH Titration
N-(2-Hydroxyethyl)diethylenetriamine trihydrochloride (L1 · 3HCl) (Fig. 1)
was prepared by a modified literature method (14). The other reagents used were
of analytical grade. The buffers, MES (4-morpholinoethanesulfonic acid, Fluka)
and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, FARCO), were
commercially available and used without further purification.
1H NMR spectra were recorded on a Bruker AM-200 spectrometer. IR spec-
tra were recorded on a Nicolet 170SXFT spectrophotometer (as KBr discs) and
microanalyses of C, H and N were determined by a Perkin-Elmer 240C elemental
analyzer.
Preparation of L1 · 3HCl
2-Chloroethanol (48.3 g, 0.60 mol), diethylenetriamine (61.9 g, 0.60 mol)
and H2O (60 mL) were added successively with stirring. The mixture was refluxed
for 8 h, and then cooled to room temperature. Methanol (75 mL) and K2CO3
(51 g, 0.37 mol) were added to the reaction mixture and the resulted precipitate
of KCl was filtered off. After the filtrate was freed of methanol, it was distilled
under reduced pressure, bp 153–155◦C (3 mm Hg). The trihydrochloride was
obtained upon bubbling dry HCl into a CH2Cl2 solution of L1, yield 8.3 g (54%),
mp 172–174◦C. Anal. Calcd. for C6H17N3O · 3HCl (M = 256.60): C, 28.07; H,
7.86; N, 16.38%. Found: C, 27.84; H, 7.70; N, 16.08%. IR (cm−1, KBr): 3432 br
–
(ν (OH)); 2799 m (δ(NH2)), 1626 m, 1454 m (δ(NH2)); 1066 w (ν(C N)); 1025
1
–
w (ν(C O)). H NMR (ppm, δ (D2O), TMS): 3.80 (2H, m, CH2OH); 3.38 (8H, m,
(CH2)2NH(CH2)2); 3.22 (2H, m, NHCH2).
Preparation of HL · 3HCl
To a solution of N-(2-hydroxyethyl)diethylenetriamine trihydrochloride
(L1 · 3HCl) (5.13 g, 0.02 mol) in ethanol (200 mL), salicylaldehyde (2.44 g, 0.02
mol) in ethanol (100 mL) was added dropwise with stirring. After stirring for ca.
2 h at room temperature, NaBH4 (0.76 g, 0.02 mol) in ethanol (100 mL) was added.
After stirring for another 2 h, HCl (5 mL, 6 M) was added to destroy excessive
NaBH4, and then the pH of the solution was adjusted to ca. 8. After it was evap-
orated effectively to dryness in vacuo, the residue was dissolved in H2O (50 mL)
and extracted five times with CHCl3 (50 mL). The organic extracts were com-
bined, washed with saturated Na2CO3 aqueous solution (50 mL), and then dried

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XIE ET AL.
over Na2SO4. Na2SO4 used was filtered off; the filtrate was evaporated to dryness
in vacuo and the product was converted into its trihydrochloride as mentioned
above, yield 5.4 g (74%), mp 233–234◦C. Anal. Calcd. for C13H23N3O2 · 3HCl
(M = 362.73): C, 43.03; H, 7.23; N, 11.59%. Found: C, 42.95; H, 7.24; N, 11.45%.
IR (cm−1, KBr): 3399 br (ν(OH)); 2783 m (δ(NH2)); 1266 m (ν(C O)); 1072 m
–
1
–
(ν(C N)); 1600 s, 762 s (δ(Ar-H)). H NMR (ppm, δ (D2O), TMS): 6.88–7.29
(4H, m, Ar); 4.22 (2H, s, ArCH2); 3.77 (2H, m, CH2OH); 3.44 (8H, s, NH(CH2)2);
3.20 (2H, m, NHCH2).
Preparation of Zn(II) Complex with HL
HL · 3HCl (0.36 g, 1.00 mmol) suspended in methanol (50 mL) was added to
Zn(ClO4)2 · 6H2O (0.37 g, 1.00 mmol) in methanol (50 mL), and then an aqueous
NaOH solution (1 mL, 5 M) was added to the reaction solution. After the mixture
was refluxed for 2 h, most of the methanol was removed by a rotary evaporator to
produce a white solid. It was filtered and washed successively with water, methanol
and diethyl ether, and then dried in vacuo, yield 0.26 g (82%), d.p. 211–213◦C.
Anal. Calcd. for C13H21N3O2Zn (M = 316.71): C, 49.30; H, 6.68; N, 13.27%.
Found: C, 48.94; H, 6.54; N, 12.97%. IR (cm−1, KBr): 3287 s (ν(N H)); 3251
–
–
–
–
m (ν(N H)); 1277 m (ν(C O)); 1083 m (ν(C N)); 1598 m, 758 s (δ(Ar)); 527 m
–
–
(ν(Zn O)); 467 m (ν(Zn N)).
pH Potentiometric Titration
Standardization of KOH, HNO3 and Zn(NO3)2 solutions and procedures of
potentiometric pH-titration together with the nonlinear fitting program used to
calculate the protonation and Zn(II) complexation constants employed the same
methods as reported (3). Protonation equilibria were studied at an ionic strength of
0.10 M KNO3 and at the ligand concentration 1.0 × 10−3 M at 25◦C. The complex
formation was studied by titrating 1.0 × 10−3 M ligand and equimolar Zn(NO3)2
at 25◦C with 0.1314 M standardized KOH solution.
Kinetics
A kinetics study was carried out on a Shimadzu UV-240 spectrophoto-
meter equipped with a thermostated cell (298 0.1 K). The hydrolysis rate of
4-nitrophenyl acetate was measured by an initial slope method following the in-
crease of the absorption at 400 nm of the released 4-nitrophenolate. The reaction
solution was maintained at 298 0.1 K and the ionic strength was adjusted to 0.10
with KNO3. Buffers (50 mmol/L) were used to maintain the pH (HEPES 7–8,
–
MES 8–9). To obtain a homogeneous solution, CH3CN H2O (10% v/v) was used

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LINEAR PENTADENTATE LIGAND
199
as a solvent. For the initial rate determination, the following typical procedure was
employed. After 4-nitrophenyl acetate and the complex solutions at a certain pH
(with the reference solution containing no complex) were mixed, the UV absorp-
tion at 400 nm was recorded immediately at 5 sec intervals. The initial slope (<5%
conversion) of a plot of the absorbance vs. time was determined (linear correlation
coefficient >0.99). All experiments were run in triplicate and the average value of
the parallel experiments was obtained as the result.
RESULTS AND DISCUSSION
Protonation and Complexation
Figure 2 A illustrates the titration curve for HL. The value of a is the ratio
of moles of base to the moles of HL.
In the titrating pH range, four protonation equilibria can be seen correspond-
ing to the phenolate and the three amino groups. The conjugate base of HL with
the phenol deprotonated can be symbolized as L−. The protonation constants β01h
and K01h are defined as following:
h−1
L− + hH+ HhL
ꢀ
β01h = [HhLh−1]/([L−][H+]h)
(h = 1, 2, 3 and 4) (1)
HhLh−1
Hh−1Lh−2 + H+ K01h = [H+][Hh−1 Lh−2]/[HhLh−1
]
ꢀ
(h = 1, 2, 3 and 4) (2)
The values obtained for log β010 − log β014 are 10.55(2), 19.78(3), 27.80(3) and
31.86(5), respectively. The pK011 − pK014 values are 10.55, 9.23, 8.02 and 4.06,
Figure 2. Titration curves for HL · 3HCl and its Zn(II) complex A) 1 mmol/L HL · 3HCl;
B) A + 1 mmol/L Zn(NO3)2.

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XIE ET AL.
respectively, where the first value corresponds to protonation of the phenolate and
the next three correspond to that of the amino nitrogens.
Curve B in Figure 2 shows the titration curve for the complexation of HL
with equimolar Zn(NO3)2. From the tiration curve it was shown that the complex
began to form at pH 4. Five protons are dissociated upon coordination with Zn2+
and the data were treated as the following equation:
h+1
Zn2+ + L− + hH+
ZnHhL
β11n = [ZnHhLh+1]/([Zn2+][L−][H+]h)
ꢀ
(3)
The values obtained for log β111, log β110 and log β11−1 are 17.88(8), 11.89
(3) and 2.72(6), respectively. Upon coordination, the phenol and alkoxyl groups
are activated and deprotonate, as represented by the following equations:
+
+
ZnHL2+
ZnL + H
Ka = [ZnL+][H+]/[ZnHL2+] = β110/β111
ꢀ
1
(4)
ZnL+
ZnH−1L + H+
Ka = [ZnH−1L][H+]/[ZnL+] = β11−1/β110
ꢀ
2
(5)
where ZnH−1L denotes that the coordinated alcoholic OH deprotonates to yield
Zn(II)· · ·−OR. The distribution diagram for this system as a function of pH is
displayed in Figure 3. The obtained values of pKa and pKa are 5.99 and 9.17,
1
2
respectively.
Figure 3. Distribution curves of Zn(II) complexes of HL · 3HCl at 25 0.1◦C, I = 0.10
mol/L KNO3.

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201
Kinetics
The hydrolysis process may be represented as the following equation:
O
CH3C
O
NO2 + H2O
(NA)
O
ZnH-1L
CH3C OH + HO
NO2
(NP)
υ = −d[NA]/dt = d[NP]/dt = dA/εdt
where A and ε represent optical density and molar extinction coefficient, respec-
tively. The following kinetic equation is obtained by a method similar to the liter-
ature (3):
υ = (kc[Zn(II)] + kOH[OH−])[NA] + k0
where kc, kOH and k0 are the catalytic rate constants of the zinc(II) complex, OH−
and water, respectively. The obtained kOH and k0 values are:
k0 = 1.14 × 10−5s−1
,
kOH = 7.85 M−1 · s−1
.
Figure 4. Rate-pH profile for the second-order rate constants of NA hydrolysis catalyzed
by Zn(II) complexes of HL · 3HCl at 25 0.1◦C, and I = 0.01 mol/L KNO3 in CH3CN
(10% v/v).

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XIE ET AL.
The kOH value is nearly the same as the literature value (3). When kc is
plotted versus pH (Fig. 4), it is not a typical sigmoid curve.
As is shown in Figure 3, [(ZnL)+] increases with the increase of pH until it
reaches a maximum at pH 7.5, and then it begins to decease. On the other hand,
[ZnH−1L] increases continuously. kc has the same trend as [ZnH−1L], indicating
that the catalysis is predominantly contributed not by (ZnL)+ but by ZnH−1L. It is
indicative of a kinetic process controlled by an acid-base equilibrium (due to the
deprotonation of the alkoxyl proton).
The main differences between (ZnL)+ and ZnH−1L is summarized as fol-
lowing: the phenol in (ZnL)+ coordinates to Zn(II), yielding Zn···−OAr, and the
coordinated phenol deprotonates with a pKa value of 5.99, while the alkoxyl in
ZnH−1L coordinates more weakly to Zn(II), yielding a nucleophile, Zn···−OR.
5
Similar to that of alcohol-pendant [12]aneN3 , the mechanism of the hydrol-
ysis of NA ester catalyzed by ZnH−1L may be tentatively proposed as
following:
The nucleophile Zn···−OR attacks the carbonyl of the NA ester, liberat-
ing a 4-nitrophenolate anion and yielding an acetyl intermediate A. The previ-
ously coordinated alkoxyl has combined with the acetyl, leaving a vacant site on
Zn(II) ion, which is then occupied by a water molecule, yielding Zn···−OH. There-
after, Zn···−OH acts as a nucleophile to attack the carbonyl of the acetyl group,
completing the catalytic cycle. As shown in Figure 4, kc reaches 8.5 ×
O
O
H3C
O
CH3
OH
O
O
−O
NO
2
NH
N
NH
Zn
NO2
Zn
O
slow
H
O
N
H
NH
NH
(A)
fast
-CH3COOH
Figure 5. Proposed mechanism of NA hydrolysis catalysed by ZnH−1L.

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203
10−2 mol−1 · L · s−1 at pH 9.04, suggesting good catalysis contributed by Zn···−OR
(pKa = 9.17). Similarly, in alkaline phosphatase (AP), the Zn(II)-bound alkoxyl
of serine residue was activated to deprotonate (pKa ca. 7.5), yielding the good nu-
cleophile Zn···−OR, which attacks the phosphate substrates. As described above,
(ZnL)+ is a much poorer catalyst than ZnH−1L, and, therefore, it is reasonable
to say that Zn···−OAr is a poorer nucleophile than Zn···−OR. This is probably
related to the difficulty for the strongly coordinated −OAr to leave the Zn(II) ion
and to attack the substrate. As seen from the above results, the nucleophiles orig-
inate from the deprotonation of suitable groups coordinated to Zn(II). The pKa
value is essential to its catalysis. It must not be too low, otherwise the nucleophile
generated may be too weak to give good catalysis. On the other hand, if the pKa
value is too large, the concentration of the nucleophile may be too low, and also
only poor catalysis will be obtained. This subtle relationship between the strength
of coordination (i.e. pKa value) and its catalysis must be considered in designing
the complexes mimicking the structures and activities of metallohydrolases.
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Received January 5, 2000
Accepted August 31, 2000
Referee I: Y. Masuda
Referee II: K. Sakata

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