Zinc(II) tweezers containing artificial peptides mimicking the active site of phosphotriesterase: The catalyzed hydrolysis of the toxic organophosphate parathion

Article abstract of DOI:10.1016/j.jinorgbio.2010.07.009

Two new ligand-containing histidine based on N,N',N"-tris(N-benzyl-l-histidinyl)tri(2-aminoethyl)amine, L¹, namely N,N',N"-tris[(1S)-2-methoxy-2-oxy-1-(1-benzylimidazol-4-ylmethyl)]nitrilotriacetamide L² and N,N',N"-tris{N-benzyl-N-[

Full text of DOI:10.1016/j.jinorgbio.2010.07.009

Journal of Inorganic Biochemistry 104 (2010) 1195–1204
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Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Zinc(II) tweezers containing artificial peptides mimicking the active site
of phosphotriesterase: The catalyzed hydrolysis of the toxic
organophosphate parathion
b,c
Mohamed M. Ibrahim a,c, , Gaber A.M. Mersal
⁎
a
Chemistry Department, Faculty of Science, Kafr El-Sheikh University, Kafr El-Sheikh 33516, Egypt
Chemistry Department, Faculty of Science, South Valley University, Qena, Egypt
Chemistry Department, Faculty of Science, Taif University, 888 Hawaiya, Saudi Arabia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 May 2010
Received in revised form 14 July 2010
Accepted 15 July 2010
Available online 23 July 2010
Two new ligand-containing histidine based on N,N′,N″-tris(N-benzyl-L-histidinyl)tri(2-aminoethyl)amine,
L1, namely N,N′,N″-tris[(1S)-2-methoxy-2-oxy-1-(1-benzylimidazol-4-ylmethyl)]nitrilotriacetamide L2 and
N,N′,N″-tris{N-benzyl-N-[N-benzyl-N-(N-benzyl-L-histidinyl)-L-histidinyl]-L-histidinyl}tri(2-aminoethyl)
amine L3 were prepared. Zinc(II) binding studies by these ligand systems were analyzed by means of
potentiometric and 1H NMR titrations in aqueous methanol (33 % v/v). Subsequently their zinc(II) complexes
[L1Zn(H2O)](ClO4)2·HClO4 (1), [L2Zn(OH2)](ClO4)2·H2O (2), and ([L3Zn3(H2O)3](ClO4)6·3HClO4·5H2O (3),
respectively were synthesized and characterized. The reactivity of the trinuclear complex (3) toward the
hydrolysis of the toxic organophosphate parathion was investigated and compared with that of the
mononuclear reference complex (1). From the pH dependence of the apparent rate constants, and the
deprotonation constant (pKa) of the coordinated water molecules in (1), the active species were confirmed
to be {[HL1Zn(OH)]2+/[L1Zn(H2O)]2+} at pH 8.5. The trizinc complex (3) effects hydrolysis of parathion, with
three times rate enhancement over the mononuclear (1), indicating that cooperative action of the three zinc
centers is limited.
Keywords:
Syntheses
Artificial peptides
Trinuclear zinc(II) complex
Modeling
Parathion
Detoxification
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
it is quite clear that the desired acceleration cannot be reached with
simple mononuclear complexes [37–44].
The development of metal-containing catalysts for the rapid
hydrolysis of toxic organophosphorus triesters is of fundamental and
practical interest since these compounds appear from day-to-day
applications as pesticides and potent chemical warfare agents [1,2].
Several hydrolytic enzymes in nature, including phosphatases [3–5],
often possess two (or more) metal ions at their active sites. These metal
ions, and/or the water bound to them, cooperate and as a result,
hydrolyze stable phosphoesters under physiological conditions [6–23].
The presence of two or three zinc(II) centers in a single complex appears
to be important for activation and recognition of cleavage sites within
phosphate esters [24–28]. Moreover, these complexes generate highly
cationic species that favor the electrostatic attraction to the phosphate
backbone.
Recently, in order to increase the reactivity of artificial hydrolytic
catalysts, several hydrolytic metalloenzyme models containing di-
and trinuclear zinc(II) complexes have been designed and studied to
account for or mimic the function played by the cooperativity of two
or three zinc(II) ions, as occurs in several phosphatases and nucleases
[45–59]. In these systems, we found that the multinuclear complexes
were generally more active than their corresponding mononuclear
ones. This inspired us to probe further into how molecular level effects
with respect to the structure of the ligand and the overall nature of the
bi- and trimetallic coordination complex affect the activity of the
catalyst system.
In our recent studies, we have investigated the mononuclear zinc
(II) complex (1), derived from the trihistidine, N,N′,N″-tris(N-benzyl-
L-histidinyl)tri(2-aminoethyl)amine L1 as a catalyst in the cleavage of
phosphate diester bis(p-nitrophenyl)phosphate (BNPP−) [60]. The
higher efficiency of (1) arises from the lowering the pKa value of Zn
(II)-bound water molecule. It is important to elucidate the cooper-
ation effect of three zinc(II) ions in phosphoester hydrolysis, since
three zinc ions have been suggested to participate in enzymatic
reactions [61–64]. Thus, one of the objectives of the present work is to
modify the structure of the histidine ligand L1 [60] and change the
metal-to-ligand ratio. The source of this high reactivity is the ability of
The search for simple, yet effective, synthetic metal containing
catalysts which mimic metalloenzymes and may exploit their
synergistic effects, is a significant goal in chemistry [29–32]. Several
studies have been devoted to elucidate the effect of the ligand
structure in modulating the reactivity of the metal center [33–36] but
⁎ Corresponding author. Chemistry Department, Faculty of Science, Taif University,
888 Taif, Saudi Arabia.
E-mail address: ibrahim652001@yahoo.com (M.M. Ibrahim).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.07.009

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M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
Scheme 1. The synthesis of ligand tris(Im-bzl-L-histiylmethylestermethyl)amine (L2).
the complex to activate the phosphate ester toward the nucleophilic
attack using the triple zinc(II) activation and their cooperativities.
As part of our studies in biomimetic hydrolytic zinc enzymes [39–
45,60] and with the aim to mimic the key features of the three zinc(II)
centers in these metalloenzymes, we designed and synthesized two new
ligands containing artificial tri and nonapeptide, namely N,N′,N″-tris
[(1S)-2-methoxy-2-oxy-1-(1-benzylimidazol-4-ylmethyl)]nitrilotri-
acetamide L2 (Scheme 1) and N,N′,N″-tris{N-benzyl-N-[N-benzyl-N-(N-
benzyl-L-histidinyl)-L-histidinyl]-L-histidinyl}tri(2-aminoethyl)amine
L3 (Scheme 2) and their zinc(II) complexes (Scheme 3) by affixing
histidine coordination sites with bulky substituents on the periphery of
nitrilotriacetic acid (NTA) and tris(2-aminoethyl)amine (TREN), re-
spectively. The protonation constants (pKa) values of the ligands as well
as the stability constants (logKst) and deprotonation equilibria (pKa
(H2O)) of their zinc complexes are reported. Further information about
zinc complexes of the ligands is obtained from R (=[Zn2+]o/[L]o)-
dependent 1H NMR titrations. To assess the degree of synergism
between zinc(II) ions in the trinuclear complex (3), its catalytic
Scheme 2. The synthesis of the artificial peptide tris{[tris(benzyl-im-histidyl-aminoethyl}amine (L3).

M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
1197
benzyl-L-histidinyl)tri(2-aminoethyl)amine L1 and its aqua zinc
complex (1) were prepared as previously described [60].
2.2. Syntheses
A frequent problem with the elemental analyses of tris(Im-bzl-L-
histiylmethylester-methyl)amine L2 is that the carbon values are
outside the accepted range. When this was the case here, at least one
additional analysis value (FAB+-Ms) was determined.
2.2.1. Synthesis of Im-bzl-L-histidine methyl ester I
Dry MeOH (83 mL, 2.60 mmol) was added to an ice-cold solution of
dicyclohexylcarbodiimide ( DCC, 0.59 g, 2.85 mmol), 1-hydroxybenzo-
triazole (HOBt, 0.40 g, 2.60 mmol) and N(α)-(t-butoxycarbonyl-N(π)-
benzyl-L-histidine (0.89 g, 2.60 mmol) in dimethylformide (DMF,
5 mL). pH was adjusted to 7.0 by N-methylmorpholine (NMM). The
reaction mixture was stirred at 0 °C for 2 h and at room temperature for
12 h, filtered, and evaporated to solidify which was suspended in
dichloromethane (30 ml), extracted with 10% sodium carbonate
(10 mL), dried which was dissolved in ethylacetate (EtOAc, 5 mL) and
set aside at 4 °C overnight. Further dicyclohexylurea (DCU) was then
filtered off and solvent was evaporated to give a hygroscopic powder.
Yield: 0.90 g (96%) of Boc-I. 1H NMR (CDCl3, s = singlet, m = multiplet,
br = broad): 7.36 (s, 1H; im 2′), 7.30–7.22 (m, 3H, H-b,c), 7.05–7.03
(m, 2H, H-a), 6.58 (s, 1H; im 5′), 5.83 (br, s, 1H, amide-NH), 4.93 (s,
2H, –CαH2), 4.49–4.44 (m, 1H, His-CβH) 3.57 (s, 3H, OCH3), 3.02–2.93
(m, 2H, His-CγH2), 1.34 (s, 9H, BOC). FAB+-MS: m/z: 359 [M+H]+
.
To BOC-I (0.90 g, 2.51 mmol), formic acid (5 mL, 98%) was added;
the removal of BOC group was monitored by TLC. After 15 h the formic
acid was removed in vacuo. The residue was taken in water (10 ml)
and washed with EtOAc (4×15 mL). The pH was then adjusted to
8 with solid sodium bicarbonate and extracted with EtOAc (3×20 ml).
The extracts were pooled, washed with saturated brine, dried over
sodium sulphate, and concentrated to dryness. Yield: 0.46 g (72%). 1H
NMR (CDCl3, s = singlet, m = multiplet): 7.48 (s, 1H; im 2′), 7.39–
7.27 (m, 3H, H-b,c), 7.11–7.09 (m, 2H, H-a), 6.66 (s, 1H; im 5′), 5.04 (s,
2H, –CαH2), 4.90–4.86 (m, 1H, His-CβH), 3.64 (s, 3H, OCH3), 3.13–3.10
(m, 2H, His-CγH2).
Scheme 3. The proposed structures of zinc(II) complexes (1), (2), and (3).
efficiency towards the hydrolysis of the toxic organophosphate
parathion (DENTP) (Scheme 4) was compared with the monomeric
analogue (1). These results obtained, in turn, were able to provide
certain clues to the nature of the mechanism of action of this new trizinc
nonahistidine complex.
2.2.2. Synthesis of tris(Im-bzl-L-histiylmethylestermethyl)amine L2
Nitrilotriacetic acid, NTA (0.17 g, 0.3 mmol) was added to an ice
cold solution of dicyclohexylcarbodiimide (DCC) (0.12 g, 0.57 mmol),
hydroxybenzotriazole (HOBt) (0.80 g, 0.51 mmol), and histidine
methyl ester I (0.233 g, 0.09 mmol) in DMF (8 mL) and the pH was
adjusted to 7 using N-methyl morpholine (NMM). The reaction
mixture was stirred at 0 °C for 2 h and at room temperature for 12 h,
filtered and evaporated to produce a solid which was suspended in
CH2Cl2 (30 mL), extracted with 10% aqueous sodium carbonate
(10 mL) and dried. The remaining solid was dissolved in EtOAc
(5 mL) and kept at 4 °C overnight. Dicyclohexylurea (DCU) was then
filtered off and the solvent was evaporated to give a white powder.
Yield: 0.71 g, 83%. Anal. Calcd. for C48H54N10O9·2H2O (985.92+
36.04): C, 60.62; H, 6.15; N, 14.73. Found: C, 59.39; H, 6.21; N, 14.57.
1H NMR (CD3OD:D2O, 3:1, s = singlet, m = multiplet): 7.71 (s, 3H; im
2′), 7.44–7.37 (m, 9H; H-b,c), 7.27–7.23 (m, 6H; H-a), 6.94 (s, 3H; im
5′), 5.21 (s, 6H; –CαH2), 4.81–4.79 (m, 3H; His_CβH), 3.58 (s, 9H;
OCH3), 3.47 (s, 6H; N-CH2), 3.13–3.07 (m, 6H; His-CγH2). FAB+-MS:
m/z: 915 [M+H]+, 825, 673, 628, 434, 272, 200, 91, and 81.
2. Experimental
Caution: perchlorate salts of amine ligands and their metal
complexes are potentially explosive and should be handled in small
quantities.
2.1. Materials
All reagents and solvents used were of analytical grade. N(α)-(t-
butoxycarbonyl-N(π)-benzyl-L-histidine and the organophosphate
parathion were purchased from Sigma. The ligand N,N′,N″-tris(N-
2.2.3. Synthesis of tris{[bis(benzyl-im-histidyl)aminoethyl}amine Hexa
L1 (0.538 g, 0.65 mmol) was added to an ice-cold solution of
DCC (0.44 g, 2.1 mmol), HOBt (0.30 g, 1.95 mmol), and N(α)-(t-
butoxycarbonyl-N(π)-benzyl-L-histidine (0.64 g, 1.95 mmol) in DMF
(8 mL). The pH was adjusted to 7.0 by addition of NMM. The reaction
mixture was stirred at 0 °C ice-bath for 2 h, and after at room
temperature for 15 h, filtered from the formed dicyclohexyl urea
Scheme 4. The toxic organophosphates.

1198
M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
(DCU), the solvent removed in vacuo to give a sticky solid material
which was suspended in dichloromethane (30 mL), extracted with
10% Na2CO3. The organic phase was collected and dried on Na2SO4.
The filtered clear solution was dried up, and redissolved in ethyl
acetate (EtOAc, 5 mL) and was kept at 4 °C overnight. Further DCU
was then filtered off, and the solvent was evaporated in vacuo to give
a hygroscopic powder of Boc-Hexa. Yield: 0.69 g, 59%. Yield: 0.93 g,
79%. Anal. Calc. for C99H120N22O12 (1810.18): C, 65.69; H, 6.68; N,
17.02. Found: C, 64.17; H, 7.06; N, 17.57. 1H NMR (400 MHz, CDCl3, s =
singlet, m = multiplet, br = broad): 7.50 (s, 6H, im 2′), 7.33–7.25 (m,
18H, H-b,c), 7.24–7.20 (m, 12H, H-a), 6.82 (s, 6H, im 5'), 5.90 (br, 6H,
amide-NH), 5.22 (s, –CαH2, 12H), 4.84 (m, 6H, His-CβH), 3.35–3.22 (m,
6H, –CδH2), 3.08–3.03 (m, 12H, His-CγH2), 2.86–2.83 (m, 6H, –CσH2),
1.33 (s, BOC, 54H).
Boc-Hexa (0.69 g) was dissolved in trifluoroacetic acid, TFA (5 mL)
and stirred for 0.5 h at room temperature, then TFA was removed in
vacuo. The remaining sticky material was dissolved in 10 mL water
and was extracted with 30 mL ethyl acetate. The aqueous phase
was collected which was then dried to obtain a hygroscopic powder
of the acidic form of Hexa-TFA. Yield: 0.72 g, 68%. Anal. Calc. for
Aqueous methanolic solution of Zn(ClO4)2⋅6H2O (30 mg, 0.08 mmol)
was then added. A white precipitate was produced, filtered off by
membrane filter, washed out by ether, and dried up in vacuo. Yield:
43 mg, 73%: Anal. Calcd. For C48H58Cl2N10O19Zn ([L2Zn(OH2)]
(ClO4)2 H2O) (1197.31+18.02: C, 47.44; H, 4.81; N, 11.53; Cl, 5.83%.
Found: C, 47.34; H, 4.92; N, 11.35; Cl, 5.79%. 1H NMR (CD3OD:D2O, 3:1,
v/v, s = singlet, m = multiplet): 7.83 (s, 2H; im 2′), 7.49–7.42 (m, 6H;
H-b,c), 7.33–7.25 (m, 4H; H-a), 7.07 (s, 4H; im 5′), 5.22 (s, 4H; –CαH2),
4.83–4.81 (m, 2H; His_CβH), 3.66 (s, 6H; OCH3), 3.56 (s, 4H; N-CH2),
and 3.18–3.10 ppm (m, 4H; His-CγH2).
2.2.6. Synthesis of the trinuclear zinc(II) complex (3)
The ligand L3 (82 mg, 0.10 mmol) was dissolved in 2 ml MeOH:
water (50%, v/v) and the pH was adjusted to 7.0 using 1.0 M HNO3.
Aqueous methanolic solution of Zn(ClO4)2·6H2O (37 mg, 0.10 mmol)
was then added. A white precipitate was produced, filtered off by
membrane filter, washed out by ether, and dried up in vacuo. Yield:
60 mg, 68%: Anal. Calc. for C123H154N31O53Cl9Zn3 ([L3Zn3(H2O)3]
(ClO4)6·3HClO4·5H2O) (3039.52+301.38+90.08): C, 43.07; H, 4.53;
Cl, 9.30; N, 12.65; Zn, 5.71%. Found: C, 42.45; H, 4.62; Cl, 9.25; N, 12.76;
Zn, 5.76%. 1H NMR (400 MHz, D2O/DMSO-d6 20:1, s = singlet, m =
multiplet): 7.73 (s, 9H, im 2′), 7.55–7.18 (m, 27H, H-b,c), 7.33–7.21
(m, 18H, H-a), 6.89 (s, 9H, im 5'), 5.27–5.18 (s, 18H, –CαH2), 4.64
(m, 9H, His-CβH), 3.28–3.15 (m, 6H, –CδH2), 3.08–2.96 (m, 6H, –CσH2),
2.54–2.43 (m, 18H, His-CγH2).
C
84H96N22O6·10CF3COOH·8H2O (1509.8+1140.2+144): C, 44.70;
H, 4.40; N, 11.03. Found: C, 44.90; H, 4.12; N, 10.53. 1H NMR
(400 MHz, D2O, s = singlet, m = multiplet: 8.79 (s, 6H, im 2′), 7.48–
7.37 (m, 18H, H-b,c), 7.35–7.30 (m, 12H, H-a), 7.27 (s, 6H, im 5'), 5.35
(s, 12H, –CαH2), 4.56 (m, 6H, His-CβH), 4.42 –4.37 (m, 6H, –CδH2), 3.49
(m, 6H, –CσH2 ), 3.18–3.05 (m, 12H, His-CγH2).
Hexa-TFA (0.70 g, 0.25 mmol) was passed through the anion-
exchange column (Dowex, OH-form) according to a procedure
described earlier for the synthesis of Hexa. Yield: 0.18 g, 53%. Anal.
Calc. for C84H96N22O6·3H2O (1509.83+54.05): C, 64.51; H 6.57; N,
19.70. Found: C, 64.99; H, 6.83; N, 19.24. 1H NMR (400 MHz, D2O, s =
singlet, m = multiplet): 7.63 (s, 6H, im 2′), 7.40–7.30 (m, 18H, H-b,c),
7.25–7.20 (m, 12H, H-a), 6.87 (s, 6H, im 5'), 5.07 (s, 12H, –CαH2), 4.50
(m, 6H, His-CβH), 3.05 (m, 6H, –CδH2), 2.89 (m, 6H, –CσH2), 2.39 (m,
12H, His-CγH2).
2.3. Potentiometric measurements
Potentiometric titrations were carried out at 25.0 0.2 °C with a
TOA AUT-501 automatic titrator connected to a TOA ABT-511
automatic burette with a combined glass electrode. The electrode
was calibrated using standard aqueous buffers. The ionic strength was
adjusted to 0.1 M by adding appropriate amounts of NaNO3. Solutions
were made up with aqueous methanol (33%, v/v) and the ionic
strength was adjusted to 0.1 M by adding appropriate amounts of
NaNO3. The glass electrode was calibrated and checked for linearity
with standard borax and potassium hydrogen phthalate as well as
acetate buffers. Checks for the presence of carbonate were made using
a Gran plot and the determination of Kw for the solvent system.
Although a correction was not made to compensate for the methanol–
water liquid junction potential a correction of 0.136 pH units can be
subtracted from the measured pH readings as suggested by Bates et al
[65] to enable a comparison to be made with measurements in
aqueous solution. All solutions were carefully protected from air by a
stream of nitrogen. Solutions of 1.00 mM of L1 in the absence and in
the presence of equivalent Zn2+ ion were titrated with 0.1 M NaOH
aqueous solution. Equilibrium constants were calculated using the
program BEST [66] and species distributions were calculated using the
program SPE [66].
2.2.4. Synthesis of tris{[tris(benzyl-im-histidyl)aminoethyl}amine L3
Hexa (0.76 g, 0.50 mmol) was added to an ice-cold solution of
DCC (0.36 g, 1.72 mmol), HOBt (0.23 g, 1.50 mmol), and N(α)-(t-
butoxycarbonyl-N(π)-benzyl-L-histidine (0.50 g, 1.50 mmol) in DMF
(8 mL). The reaction mixture was worked up in a similar fashion as
described for the synthesis of Boc-Hexa.
The protected BOC-L3: Yield: 0.74 g, 49%. C138H156N31O15 (2488.96):
C, 66.59; H, 6.32; N, 17.45. Found: C, 65.04; H, 6.53; N, 17.93. 1H NMR
(400 MHz, CDCl3, s = singlet, m = multiplet, br = broad): 7.41 (s, 9H,
im 2′), 7.30–7.24 (m, 27H, H-b,c), 7.22–7.18 (m, 18, aromatic, H-a), 6.79
(s, 9H, im 5'), 5.91 (br, s, 9H, amide-NH), 5.24 (s, 18H, –CαH2), 4.66 (m,
9H, His-CβH), 3.39–3.28 (m, 6H, –CδH2), 3.12–3.07 (m, 18H, His-CγH2),
2.88–2.81 (m, 6H, –CσH2 ), 1.42 (s, Boc, 81H).
The acidified L3-TFA: Yield: 0.77 g, 69%: Anal. Calc. for C123H132N31O9·
13CF3COOH·5H2O (2191.6+1482.3+90): C, 44.03; H, 3.92; N, 10.68.
Found C, 44.65; H, 3.84; N, 10.57. 1H NMR (400 MHz, D2O, s = singlet,
m = multiplet): 8.68 (s, 9H, im 2′), 7.52–7.43 (m, 27H, H-b,c), 7.33–7.27
(m, 18H, H-a), 7.22 (s, 9H, im 5'), 5.40 (s, 18H, –CαH2), 4.61 (m, 9H, His-
CγH), 4.41–4.37 (m, 6H, –CδH2), 3.48 (m, 6H, –CσH2 ), 3.20–3.09 (m, 18H,
His-CγH2).
2.4. 1H NMR measurements
To confirm stoichiometry of zinc complexes of each ligand, 1H
NMR analyses were undertaken in order to investigate the effect of pH
and the added zinc on the shape and the chemical shift of ligand
peaks. From the pH or R (=[Zn2+]o/[L]o)-dependence of chemical
shifts, we can get more insight about that which protons are effected
(thus shifted) most, i.e. gives evidence about the location of
protonation/complexation sites [45,67]. The concentrations used
were analogous to that was used in pH-titrations; ionic strength
was kept constant at 0.1 M by using NaNO3. D2O was used as solvent
for the titration of L1, while in case of L3, a solution of MeOH-d4 and
D2O (33% v/v) was used. The concentration of the ligands (2×10−3 M)
in D2O and I=0.1 M NaNO3 was kept constant at a pH≈7 at 30 °C.
Chemical shifts are reported relative to the resonance signal of sodium
2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as internal standard. The
The ligand L3: Yield: 0.26 g, 57%: Anal. Calc. for C123H132N31O9·4H2O
(3199.57+72.07): C, 65.26; H, 6.37; N 19.18. Found: C, 66.76; H, 6.01; N,
19.55. 1H NMR (400 MHz, D2O, s = singlet, m = multiplet): 7.64 (s, 9H,
im 2′), 7.39–7.33 (m, 27H, H-b,c), 7.29–7.22 (m, 18H, H-a), 6.84 (s, 9H,
im 5'), 5.12 (s, 18H, –CαH2), 4.46 (m, 9H, His-CγH), 3.09 (m, 6H, –CδH2),
2.88 (m, 6H, –CσH2), 2.37 (m, 18H, His-CγH2).
2.2.5. Synthesis of the mononuclear zinc(II) complex (2)
The ligand L2 (50 mg, 0.04 mmol) was dissolved in 3 mL aqueous
metanol (75%, v/v) and the pH was adjusted to 7.0 using 1.0 M HNO3.

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1199
pD was adjusted with concentrated NaOD and DNO3, so that the effect of
dilution could be neglected.
2.5. Hydrolysis reaction of the toxic organophosphate, parathion
The hydrolysis rate of parathion, DNTP catalyzed by Zn2+-bound
hydroxo species of (1), (2), and (3) in aqueous media was measured
following an increase in 402 nm absorption of released p-nitrophenolate
as a hydrolysis product [39–45]. Application of co-solvent was necessary
in the case of L2 and L3 (MeOH:H2O, 33%, v/v): reactions with L1 have
been carried out in both H2O and MeOH:H2O (33%, v/v). The hydrolysis
rate of parathion, DENTP (4.0×10−5 M) catalyzed by their zinc(II)
complexes (1)–(3) (1.0×10−3 M) in aqueous methanol was measured
following an increase in 402 nm absorption of released p-nitrophenolate
as a hydrolysis product. The pH of each sample was adjusted by using 1 M
NaOH/HNO3, and not by using buffers in order to avoid any catalytic or
inhibition effect. In the course of hydrolysis, the pH of each sample was
checked and was found that the difference compared to the adjusted pH
value at the beginning of hydrolysis was not larger than 0.3. The samples,
included the organophosphate and both complexes were incubated at
30 °C, while the applied parathion concentration was 20 μM. All reactions
showed first-order characteristics and were followed up to 5 half lives.
Reaction rates are corrected by blank experiments which were made up
similarly but without the presence of the zinc complexes. The pseudo-
first order rate constants, as before, were estimated from the integrated
form of the first-order law.
Fig. 1. Distribution curve of zinc-containing species of 1.0×10−3
M
of L1 in the
presence of equivalent amount of Zn2+ ions in aqueous methanol (33%, v/v) at I=0.1 M
NaNO3 and 30 °C.
evaluate the pH-metric curves. The obtained deprotonation constants
pKa are summarized in Table 1. In ligand L2, three consecutive
deprotonation steps were observed and assigned to the three
imidazole nitrogens (Table 1). In dipeptides, where the amino group
of histidine is unprotected, the pKa of the imidazole nitrogens is lower
than in histidine: e.g. 5.39 in His-Gly, 5.65 in His-Leu while this value
is 5.87 in histidine [68,69]. Thus, L2 has more basic imidazole
nitrogens [70,71] because it lacks other aliphatic nitrogens, which is
found in L1 [60,72–74], where the presence of these aliphatic amines
makes the imidazol groups more acidic.
3. Results and discussion
3.1. Characterization of the ligands and their zinc(II) model complexes
The general aim of the synthesis of these artificial peptides is to
mimic the structure of the phosphohydrolase active site by not only
utilizing zinc as natural ion in zinc-containing enzymes, but also to
incorporate peptides as the building blocks of large proteins. The
increasing number of the attached histidyl units will come together
with the formation of multinuclear zinc complexes. The benzylation of
pyrrolic imidazole nitrogen in these ligands L1, L2, and L3 protects the
imidazole ring of histidine from side reactions during synthesis and
later will have an important role in zinc coordination since only the
pyridine-type nitrogen of the imidazole ring will be available for
coordination. The three histidyl residues coupled to nitrilotriacetic
acid (NTA) in L2 and tris(2-aminoethyl)amine (TREN) in L1 and L3 via
amide bond formations mimicked the histidine side chain in large
proteins, which is common in the active site of several zinc enzymes.
We tried to attach benzyl groups to each histidyl unit in order to:
(i) increase the hydrophobicity around zinc, (ii) avoid octahedral
dimer formation by introducing benzyl groups as a steric hindrance,
and (iii) help binding of aromatic substrates by π–π stacking. The
advantageous of these ligands can be summarized in the following
statements: (i) the donation sites of imidazoles have kinetically more
labile binding ability, (ii) there is a larger spacer distance between the
imidazole donor set and the tertiary nitrogen, (iii) there is an
increased flexibility for coordination thus forming a more optimal
geometry, and (iv) for steric reasons, it is impossible to bind the
central tertiary nitrogen.
The potentiometric pH titration curve for the ligands L1 and L2 in
the presence of equimolar Zn2+ revealed complex formations until
[OH]/[L]=7.0 [61] and 3.0, respectively. But in all cases Zn complexes
reacted with more OH− than what was needed to fully deprotonate
the ligands. This means each complex contains one extra deproton-
able group which is not present in the ligands themselves. In the case
of complex (2), the extra deprotonation can only be assigned to the
deprotonation of water molecule ligated to the central zinc ion of the
complex. In the case of complex 1, the extra deprotonation could be
assigned to two different processes: (i) deprotonation of the
coordinated water molecule or (ii) metal-promoted deprotonation
of the amide. Rabenstein et al. [67] showed that zinc(II)-promoted
deprotonation of amide nitrogens in imidazole-containing peptides
generally results in kinetically stable complexes. The chemical shifts
of the protons near to the amide nitrogen are considerably altered. In
3.1.1. Deprotonation and zinc complexation constants of L1 and L2
The obtained ligands: L1, L2, and L3 provided an opportunity to
examine the effect of changing the number of histidine side chains on
the ligand deprotonation and complexation constants, and neverthe-
less on the pKa of the coordinated water molecule. Typical set of
species distribution curves for L1 and L2 in the presence of
stoichiometric amounts of Zn2+ are shown in Figs. 1 and 2. While in
case of ligand L3, the number of ligation sites far exceeds the capacity
of the fitting program, so no attempts were made to measure and to
Fig. 2. Distribution curve of zinc-containing species of 1.0×10−3
M
of L2 in the
presence of equivalent amount of Zn2+ ions in aqueous methanol (33%, v/v) at I=0.1 M
NaNO3 and 30 °C.

1200
M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
Table 1
Table 2
Stability constants (logKst) and deprotonation constants (pKa(H2O) of L2 and its zinc(II)
Stability constant (logKst) and deprotonation constant (pKa(H2O) of the indicated
species of zinc(II) complex 2.
complex species.
Species/reactions
pKa
logKst
pKa (H2O)
Species/reaction
LogKst
pKa (H2O)
HL+ =L+H+
5.78
5.23
4.36
L2 +Zn2+ =L2Zn2+
3.95
3.95
7.33
H2L+ =HL+ +H+
H3L3+ =H2L2+ +H+
L+Zn2+ =LZnOH+2
LZnOH+2 =LZnOH+ +H+
L2ZnOH22 =L2ZnOH2+ +H+
3.95
7.33
The monoprotonated complex species, [HL1Zn(H2O)]3+ contains
zinc-bound water molecule and one protonated primary amino group.
The proton loss of this species can occur either at the amino function
to form [L1Zn(H2O)]2+ (pKa =8.38) or at the coordinated water
molecule to form [HL1Zn(OH)]2+ (pKa =7.43). As these species are
titrimetrically equivalent, it is not possible to distinguish between
order to clarify whether the amide nitrogens are coordinated to zinc
or not, we have conducted 1H NMR titration measurements for ligand
L1 (2.0×10−3 M) in the presence of equimolar amount of Zn2+ ion in
D2O (Fig. 3) as a function of pD (above pD 6.0). Through this, we found
out that the methylene protons of the TREN part (β), directly linked to
the amide nitrogens were weakly influenced (0.03 ppm). The above
result strongly indicates that the extra deprotonation is only related to
the formation of zinc-bound hydroxo species and not with metal-
promoted deprotonation of amide nitrogens.
The obtained stability constants logKst and deprotonation con-
stants pKa(H2O) of zinc complexes (1) and (2) are listed in Table 2.
The potentiometric data indicated that L2 forms a less stable complex
with Zn(II) (logKst =3.95). The lowering in logKst value of (2) is
consistent with related complexes bearing only 3 imidazole donor
functions [70,71] and is consistent with the protonated species
[H3L1Zn]5+ of the ligand L1. The lowering pKa(H2O) in both
complexes: (1) (7.43), and (2) (7.33) may arise because of the
bulkiness around each coordinated water molecule. N-alkylation of
the pyrrolic nitrogen in imidazole units reduces the metal binding
ability of the ligand. For example in the case of tripodal N-methyl
imidazole derivatives, logKst decreases about 3–4 units, with a logKst
value of around 4, which is consistent with the analogous value of
[H3L1Zn]5+ species [70,71]. Subsequent deprotonation of aliphatic
amino groups forms complexes with increasing logkst values
indicating some contribution of amino nitrogens in coordination
with the central zinc ion.
them by pH-metric titration. Since the stability of [HL1Zn(OH)]2+
/
[L1Zn(H2O)]2+ is not much higher compared to [HL1Zn(H2O)]3+, it
indicates that [HL1Zn(OH)]2+/[L1Zn(H2O)]2+ contains no new coor-
dination site in comparison with [HL1Zn(H2O)]3+. A possible newly
formed binding site would probably contribute to ligate the central
zinc, resulting in higher logkst values. If the [HL1Zn(H2O)]2+ is formed,
there is no increase in the coordination number of the central zinc and
is not associated with dramatic change in logKst. From the above
reasons we assume that when [HL1Zn(H2O)]3+ loses a single proton,
the zinc-bound hydroxo species, rather than the [L1Zn(H2O)]2+ is
formed.
3.1.2. pD and R (=[Zn2+]o/[L]o)-dependent 1H NMR titrations
To get additional information about the pD-dependence of
complex formation between zinc and the different ligands, another
set of experiments has been performed where the zinc-to-ligand ratio
was kept constant and the pD of the solution was varied. The aromatic
region of the spectra is shown in separate charts in Fig. 4. The free
ligand L1 (R=0.0) demonstrates strong upfield shift between pD 4.0
and 6.0, especially the 2′-imidazole peak is influenced and is in
relation with the proton loss of the pyridine-type of the imidazole
nitrogens. When pD is increased, slight upfield shift for the imidazole
peaks are observed which is due to the deprotonation of the primary
amino nitrogen.
At R=1.0 there is no change found at pD 4, in comparison with the
free form at the same pD indicating that even in presence of 1.0
equivalent of zinc, no complexation takes place at this pD. At pD 5.0,
broadening of imidazole peaks indicates at least partial formation of
the mononuclear [H3L1Zn]5+ complex. At pD higher than 7.0, the
imidazole peaks show change in chemical shift and peak shape when
compared to the spectrum of the free ligand. The deprotonation
process of [HL1Zn(H2O)]3+ ↔{[HL1Zn(OH)]2++[L1Zn(H2O)]2+}+H+
was described by a pKa of 7.43 by pH-titration, but since mixture of aqua
and hydroxo species are yielded, the effect of hydroxo species formation
is hardly seen in the pD-dependent 1H NMR spectra. At pD 9.0,
broadening of all aromatic peaks is observed, and can be related to the
appearance of [L1Zn(OH)]+ species, which is present in around 20%
according to the distribution curve calculated from the pH-metric
titration.
1H NMR spectra for the R-dependence of L3 is presented in Fig. 5.
The L3 contains tripeptide units in the side chains, the number and the
ratios of different types of binding sites in this ligand makes it a special
representative of artificial histidine-containing peptides. As shown in
Fig. 5, in case of free ligand L3 ligand, all peaks are well resolved. When
increasing R from 0.0 to 1.0, significant changes in both peak positions
and peak shapes were recorded. At R=0.5, the singlet peaks of 2′ and
5′-imidazolyl protons as well as phenyl protons show broadening and
shifts downfield, which is clear indication about the fast chemical
exchange between the complexed and uncomplexed L3. At R=1.0 the
aromatic peaks show some shift. This indicates that in mononuclear
complexes only third of the 9 histidines take part in binding to zinc.
When R is increased from 1.0 to 3.0, all peaks show considerable
Fig. 3. Upfield region for the methylene protons of the TREN part (a and b) linked to the
amide nitrogens for L1 (2.0×10−3 M) in the presence of equimolar amount of Zn(II)
(2.0×10−3 M) as a function of pD in deutrated aqueous methanol: D2O:CD3OD (33%, v/v)
at I=0.1 M NaNO3 and 30 °C.

M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
1201
Fig. 4. Downfield region for the imidazole (2′ and 5′) and phenyl protons of the free ligand L1 (left) and in the presence of equimolar amount of zinc(II) (2.0×10−3 M) (right) as a
function of pD in deutrated aqueous methanol: D2O:CD3OD (33%, v/v) at I=0.1 M NaNO3 and 30 °C.
broadening and shifting downfield, and when further zinc is added, no
change is observed in peak shapes and positions. This clearly proves
that the maximum stoichiometry of zinc-L3 complex is 3:1.
have been carried out to investigate the effect of the presence of
histidine-containing peptide–zinc complexes on the hydrolysis rate of
the toxic organophosphate, parathion (DENTP). In this part we report
the results of a series of experiments carried out at different pH and
zinc-to-ligand ratios. From these profiles we make suggestion about
the active species, and from the comparison of activities of different
complexes we attempt to propose reaction mechanism and some
comments on the structure of zinc complexes are also made.
3.2. Hydrolysis of parathion by using zinc(II) model complexes (1) and (3)
The histidine units attached to the different oligoamine cores
through peptide bond model the histidine side chains of the active site
of phosphohydrolase enzymes [3–5]. The purpose of using zinc as a
complexing ion was to keep the structural similarity between the
native system and our model compounds. In order to show that these
zinc complexes do not only serve as structural mimics, kinetic studies
The catalytic activity of zinc(II) complex (1) with maximum
stoichiometry is strongly influenced by the pH of the reaction mixture.
The cleavage of parathion was followed by monitoring the increase in
the visible absorbance at 400 nm caused by release of the p-
nitrophenolate ion. The values of pseudo-first-order rate constants,
kobs (Table 2) obtained from the semilogarithmic plots for the hydrolysis
of reaction at different pH values in aqueous methanol (33%, v/v)
(Supplementary material, S1) were linear, confirming the first order
dependence on the substrate concentration. The pH-dependence of the
pseudo-first order rate constants on the hydrolysis of parathion
promoted by the zinc complex (1) with the maximum stoichiometry
(Fig. 6) displayed bell-shaped curve with the maximum rate constant of
1.36×10−5 s−1 at pH 8.5. Bell-shaped profiles (fitted by Lorentzian
function, 4 parameters) are seen in a number of phosphate ester
hydrolysis promoted by zinc complexes, and are indicative of the
involvement of the nucleophilic zinc-hydroxo [60,61,74]. This rate
profile largely correlates with the distribution curve of species {[HL1Zn
(OH)]2+/[L1Zn(H2O)]2+}. Below and above pH 8.5 the rate constants
decrease with the simultaneous decrease of the percentage of the above
mentioned species. The results of pH-metric titration suggested the
coexistence of two species with maximum abundance at pH 8.5: one is
with zinc-bound water, [L1Zn(H2O)]2+ while another is zinc-bound
hydroxo species, [HL1Zn(OH)]2+. In latter species, one of the terminal
amino group is protonated. It was not possible to distinguish between
these two species by means of pH-metric titration due to their
tautomeric structure. Ifthe aquatedspecies played role inthehydrolysis,
the zinc-bound water would not be sufficiently strong as general base,
compared to the hydroxo-species. At pH below 7.0, where only water
bound species exist as major species, a significantly diminished activity
Fig. 5. Downfield region for the imidazole (2′ and 5′) and phenyl protons of the free
ligand L3 (2.0×10−3 M) as a function of R=[Zn2+]o/[L3]o at pD 8.5 in deutrated
aqueous methanol: D2O:CD3OD (33%, v/v) at I=0.1 M NaNO3 and 30 °C.

1202
M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
Around pH 10.5 [L1Zn(OH)]+ exists in solution, while zinc-bound water
complex is absent. A question then arises as to why [L1Zn(OH)]+ is
practically inactive in this process. A probable explanation can come
from the coordination properties of this species. Since the proton loss
step of [HL1Zn(OH)]2+ occurs in one of the primary amino function, the
fully deprotonated complex contains one more ligation site, which may
also coordinate to zinc. A possible increase in coordination number of
zinc may radically change the conformation of the complex thus making
it difficult for OH− to interact with the substrate. The hydrophobic
benzyl groups attached to the imidazole binding sites can serve as
hydrophobic microenvironment but due to their bulkiness, can also
sterically hinder the approach of the substrate. In the catalytic
mechanism for the action of the species [HL1Zn(OH)]2+/[L1Zn(H2O)]2+
(Scheme 5), they play a cooperative role for the hydrolysis of parathion
through the Lewis acid mechanism. The zinc bound-OH species, acts as a
nucleophile for acceleration of the hydrolysis reaction, while water-
coordinated Zn species can supply proton as general Lewis acid holding
the substrate for OH− attack. The bound-hydroxide in [HL1Zn(OH)]2+
can attack the phosporus, and through a pentacoordinated transition
state, the leaving group p-nitrophenolate is released. The formed diethyl
thiophosphate bound to the catalyst canbe replaced by another molecule
of DENTP and the catalytic cycle can be started again. The R (=[Zn2+]o/
[L]o)-dependence study was carried out in ligand L3 to demonstrate the
effect of the degree of complexation on the catalytic activity on the
hydrolysis reaction. The applied range of R for the ligands was decided on
the basis of the R-dependent 1H NMR titration results. The values of
pseudo-first-order rate constants, kobs (Table 3) obtained from the
semilogarithmic plot for the hydrolysis of parathion at different Zn2+/L3
ratios in aqueous methanol (33%, v/v) at pH 8.5 (Supplementary
materials, S2) were linear, confirming the first order dependence on
the substrate concentration. The plot of the rate constants as a function of
zinc-to-ligand ratio (R) is shown in Fig. 7.
Fig. 6. Plots of the hydrolysis rate constant, kobs of 4.0×10−5 M parathion (DENTP) by
using zinc(II) complex (1) (1.0×10−3 M) at different pH values (● observed values and
fitted curve by Lorentzian function, 4 parameters) in aqueous methanol (33%, v/v) at
I=0.1 M NaNO3 and 30 °C.
of [L1Zn(H2O)]2+ is found which means that aqua-species solely cannot
be active catalysts for DENTP hydrolysis. In parallel with this, at pH
higher than 9.0, the percentage of aquated species is decreasing with the
concomitant decrease of the rate constants, which is clear indication
about that merely hydroxo-complex species possess lower activity than
in the presence of aquated [L1Zn(H2O)]2+. It is interesting to observe
that in the pH range 9.0−10.0, the rate constants do not fall that
drastically as the percentage of {[HL1Zn(OH)]2+/[L1Zn(H2O)]2+} spe-
cies. This may suggest that [L1Zn(H2O)]2+} also possesses some activity,
but with considerably lower value compared to what is found in the case
of [HL1Zn(OH)]2+/[L1Zn(H2O)]2+. The mechanism for the hydroxo-
complex catalyzed reaction probably follows an analogous pathway as
was discussed for [L1Zn(OH)]+ (see above). The higher activity of
[HL1Zn(H2O)]2+ can be related to the decreased pKa value of the
coordinated water molecule: the proton loss in the process of [HL1Zn
(H2O)]3+ ↔{[HL1Zn(OH)]2++H+ has a pKa of 7.68, which is signifi-
cantly lower than the pKa for [L1Zn(H2O)]2+↔[L1Zn(OH)]+ +H+
which was determined to be 8.70, thus the nucleophilicity of the bound-
hydroxide can be higher in {[HL1Zn(OH)]2+ than in [L1Zn(OH)]+. From
the pKa values of L1, HL1+ species exists around pH 7.9 and above pH 8.0
this species gradually decreases, i.e., the [HL1Zn(OH)]2+ also decreases.
Ligand L3 can form a trinuclear zinc complex which is the possible
maximum stoichiometry as it was demonstrated from 1H NMR
titrations. The R-dependence of rate constants for L3 showed
extremely low activity below R=1, even lower than the complexes
of L1. This can be explained by the higher coordination number of zinc,
more precisely the larger variety of donor groups in L3 in comparison
with the L1 (Table 4). The highly coordinated central zinc in the
mononuclear complex of L3 might reduce the nucleophilicity of the
bound-water molecule, and at the same time, may leave no sufficient
free space around the coordinated water to interact with the
phosphoester substrate. The rate constants increase sharply between
Scheme 5. The proposed mechanism for the hydrolysis of parathion by using zinc(II) complex (1).

M.M. Ibrahim, G.A.M. Mersal / Journal of Inorganic Biochemistry 104 (2010) 1195–1204
1203
Table 3
Table 4
The observed pseudo-first order rate constants (kobs (s−1)) for the cleavage of
parathion (DNTP) (4.0×10−5 M) by using zinc(II) complex (1) (1.0×10−3 M) at
different pH values in aqueous methanol (33%, v/v) at I=0.1 M NaNO3 and 30 °C.
The observed pseudo-first order rate constants (kobs (s−1)) for the cleavage of
parathion (DNTP) (4.0×10−5 M) as a function of R=[Zn2+]o/[L3]o at pH 8.5 in aqueous
methanol (33%, v/v) at I=0.1 M NaNO3 and 30 °C.
pH
106 kobs (s−1
)
R=[Zn2+]o/[L3]o
105 kobs (s−1
)
7.0
7.5
8.0
8.5
9.0
9.5
10.0
1.96
3.26
7.77
13.60
6.44
3.24
3.03
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.704
1.52
1.69
3.08
4.20
4.54
4.47
1/1 and 3/1 zinc-to-ligand ratio which indicates the high catalytic
activity of the formed trinuclear complex of L3. Since the rate
acceleration is 3-fold for L3 when considering the rate constants of
R=3 in comparison with R=1, cooperation between the catalytic
zinc centers is indicated. The rate constants level off above R=3,
which is in consistence with the 1H NMR-titration results that showed
3/1 as maximum complex stoichiometry. When ligand L3 in itself was
applied, moderate cleavage rates have been observed, smaller than
the values for the reactions catalyzed by zinc(II) complex (3). Because
of the lack of cooperation between zinc(II) ions in (1), the catalytic
activity of (3) can be thought as resulting from the sum of
contributions from three mononuclear arrangements as previously
discussed in connection with the reactivity of other trinuclear zinc(II)
complexes of calix[4]arene based ligands [12–16]. However, the
trizinc complex (3) is only ca 3–4 fold more reactive than the
mononuclear reference (1), indicating that cooperative action of the
three zinc centers is limited. This could be attributed to the non-
bridging spaceres between the three trihistidyl units (i.e., the lack or
the weak coordination of zinc ions with the tertiary nitrogen atoms of
the TREN part) produces very little pre-organization of the struc-
ture [75]. Due to the complexity of this system, it is difficult to obtain
exact analysis on this dynamic complexation system.
indeed highly reactive, producing in the case of (3) an ~25,000-fold
rate acceleration over the background reaction at pH 8.5 and 30 °C.
However, the trizinc complex (3) is only ~3 fold more reactive than
the mononuclear reference (1), indicating that cooperative action of
the three zinc centers is limited. This could be attributed to the non-
bridging spacers between the three trihistidyl moieties (i.e., the lack
or the weak coordination of zinc ions with the tertiary nitrogen atoms
of the TREN part) produces very little pre-organization of the
structure. The results obtained, in turn, were able to provide certain
clues to the nature of the mechanism of action of this new trizinc
nonahistidine complex (3). But due to the complexity of this system, it
is difficult to obtain exact analysis on this dynamic complexation
system.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jinorgbio.2010.07.009.
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