Ligand modifications modulate the mechanism of binuclear phosphatase biomimetics

Article abstract of DOI:10.1016/j.poly.2012.06.008

Complexation of dimethyl-6,6′-(2-hydroxy-5-methyl-1,3-phenylene) bis(methylene)bis((2-hydroxyethyl)azanediyl)bis(methylene)dipicolinate (Me ₂H₃L4) and 2,2′-(2-hydroxy-5-methyl-1,3-phenylene) bis(methylene)bis(((6-methylpyridin-2-yl)methyl)azanediyl)diethanol (H ₃L5) with Zn(II) afforded the complexes [Zn₂(H ₂L4)(H₂O)₂](ClO₄) and [Zn ₂(H₂L5)(CH₃CO₂)(H ₂O)](PF₆)₂·2H₂O, which were characterized by ¹H and ¹³C NMR spectroscopy, mass spectrometry, microanalysis, and the former by X-ray crystallography. Functional studies of the zinc complexes with the substrate bis(2,4-dinitrophenyl) phosphate (BDNPP) showed the complexes to be competent catalysts with k _cat = 3.52 ± 0.03 × 10^-4 and 1.27 ± 0.04 × 10^-3 s^-1 (K_m = 6.7 ± 0.9; 13.8 ± 1.5 mM), with catalytically relevant pK_as of 9.4 and 6.6, respectively. The pK_a values are discussed with respect to the potential nucleophilic species and the effect of the donor environment.

Full text of DOI:10.1016/j.poly.2012.06.008

Polyhedron 52 (2013) 1336–1343
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Ligand modifications modulate the mechanism of binuclear phosphatase
biomimetics
Kristian E. Dalle a, Lena J. Daumann a, Gerhard Schenk a,b, Ross P. McGeary a,c, Lyall R. Hanton d,
Lawrence R. Gahan a,
⇑
a School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
b Department of Chemistry, National University of Ireland – Maynooth, Maynooth, Co. Kildare, Ireland
c School of Pharmacy, The University of Queensland, Brisbane, QLD 4072, Australia
d Department of Chemistry, Te Tari Hua-Ruanuku, University of Otago, PO Box 56, Dunedin, New Zealand
a r t i c l e i n f o
a b s t r a c t
Complexation of dimethyl-6,60-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis((2-hydroxyethyl)
azanediyl)bis(methylene)dipicolinate (Me2H3L4) and 2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(meth
ylene)bis(((6-methylpyridin-2-yl)methyl)azanediyl)diethanol (H3L5) with Zn(II) afforded the complexes
[Zn2(H2L4)(H2O)2](ClO4) and [Zn2(H2L5)(CH3CO2)(H2O)](PF6)2ꢀ2H2O, which were characterized by 1H
and 13C NMR spectroscopy, mass spectrometry, microanalysis, and the former by X-ray crystallography.
Functional studies of the zinc complexes with the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP)
showed the complexes to be competent catalysts with kcat = 3.52 0.03 ꢁ 10ꢂ4 and 1.27 0.04 ꢁ
10ꢂ3 sꢂ1 (Km = 6.7 0.9; 13.8 1.5 mM), with catalytically relevant pKas of 9.4 and 6.6, respectively. The
pKa values are discussed with respect to the potential nucleophilic species and the effect of the donor
environment.
Article history:
Available online 15 June 2012
Dedicated to Alfred Werner on the 100th
Anniversary of his Nobel Prize in Chemistry
in 1913
Keywords:
Coordination Chemistry
Biomimetic
Phosphoesterase
Zinc(II) complex
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
acid (H5L4) ([Zn2(H2L4)(H2O)2](ClO4)ꢀ3H2O) and a methyl substitu-
ent, with 2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)
Organophosphorous compounds (OPs) are commonly used as
pesticides [1,2] and nerve agents [1,3,4], and act by inhibiting
acetylcholinesterase, thus preventing proper functioning of nerve
cells. OP-hydrolyzing enzymes [3–5] are thought to natively contain
one or two Zn(II) ions in the active site, although other combinations
have been suggested [6,7]. The active site of the metalloenzyme is
typically composed of a nitrogen/oxygen coordination environment
with metal ions coordinated by nitrogen donors located on histidine
ligands, oxygen donors typically from an aspartate, and often
including a metal ion-bridging hydroxide ion [8–12]. Our interest
is in model systems capable of reproducing the electronic, structural
and reactivity characteristics of OP-hydrolyzing metalloenzyme
systems [13–16]. Appropriately placed substituents, such as
hydrogen bonding groups (e.g. amines), have been shown to have
pronounced effects on reactivity in model systems [17,18], and
these also mimic the second coordination sphere effects seen in
metalloenzyme systems [19–21]. In this work we have explored
the role of a carboxylate substituent using the zinc(II) complex of
the ligand 6,60-((((2-hydroxy-5-methyl-1,3-phenylene)bis(methy-
lene))bis((2-hydroxyethyl)azanediyl))bis(methylene))dipicolinic
bis(((6-methylpyridin-2-yl)methyl)azanediyl)diethanol (H3L5) and
[Zn2(H2L5)(CH3CO2)(H2O)](PF6)2ꢀ2H2O. The catalytic potential of
both complexes has been explored using the phosphodiester
substrate bis(2,4-dinitrophenyl)phosphate (BDNPP).
2. Experimental
2.1. Materials and physical methods
Nuclear Magnetic Resonance (NMR) spectra were measured
with Bruker AV300, AV400 and AV500 instruments. The spectra
were recorded in CDCl3, CD3OD or (CD3)2SO. Chemical shifts were
determined in ppm, relative to known residual solvent peak
references. Coupling constants are given in Hz. Two-dimensional
correlation spectroscopy (COSY), heterobinuclear single quantum
correlation (HSQC), and heterobinuclear multiple bond connectiv-
ity (HMBC) experiments were used to assign each signal in the
spectra of the final ligands. Low resolution mass spectral (LRMS)
data were collected with a Bruker Esquire high capacity ion trap
electrospray ionization mass spectrometer (HCT ESI-MS), in meth-
anol (MeOH), acetonitrile (MeCN) or 50:50 MeCN:water, with a
Bruker ES source. High resolution mass spectra were obtained
using a Bruker microTOFQ ESI-MS in MeOH. The predicted isotopic
⇑
Corresponding author.
E-mail address: gahan@uq.edu.au (L.R. Gahan).
0277-5387/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.06.008

K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
1337
splitting patterns of peaks were calculated using the program
Molecular Weight Calculator [22]. UV–Vis absorption spectra were
measured with a Hitachi U3000 Spectrophotometer with 10 mm
glass or quartz cuvettes. FT-Infrared Spectroscopy was carried
out with a Perkin Elmer FT-IR Spectrometer SPECTRUM 2000, with
a Smiths DuraSamplIR II ATR diamond window. Silica gel 60 from
Merck 0.040–0.063 mm, 230–400 mesh, was employed for column
chromatography. Elemental analyses were performed using the
microanalysis facilities at The University of Queensland.
2.2.3. Synthesis of methyl-6-(2-hydroxyethylamino)methyl picolinate
Methyl-6-formylpicolinate (1 g, 6.1 mmol) was dissolved in
methanol (10 mL) and cooled to 0 °C. 2-Aminoethanol (0.37 mg,
6.1 mmol) in methanol (5 mL) was added dropwise, and the mix-
ture was stirred for two hours at room temperature. The mixture
was cooled to 0 °C, and NaBH4 (0.22 g, 6.1 mmol) was added in
small portions. After stirring for 10 min at 40 °C, water (20 mL)
was added and the mixture was concentrated to 20 mL in vacuo.
The aqueous phase was extracted with dichloromethane
(2 ꢁ 20 mL), then the aqueous phase was acidified to pH 2 and fur-
ther extracted with dichloromethane (2 ꢁ 20 mL). The combined
organic layers were dried over sodium sulfate. After removal of
the solvent, a yellow oil was obtained in 47 % yield (580 mg) 1H
NMR (CDCl3, 400.13 MHz); d 2.05 (s, 1H, NH); 2.95 (t, 2H, NCH2CH2,
J = 4.9 Hz); 3.54 (bs, 1H, OH); 3.72 (t, 2H, NCH2CH2, J = 4.9 Hz); 3.98
(s, 3H, OCH3); 4.11 (s, 2H, NCH2py); 7.56 (m, 1H, pyH, J = 4.1,
0.9 Hz); 7.84 (m, 1H, pyH); 8.00 (dt, 1H, pyH, J = 7.6, 0.6 Hz). 13C
NMR (CDCl3, 100.62 MHz); d 51.0 (CH2); 52.8 (OCH3); 53.8 (CH2);
60.4 (NCH2CH2); 123.8 (pyCH); 125.9 (pyCH); 137.7 (pyCH);
147.4 (pyCCO2Me); 160.3 (pyCCH2); 165.5 (CO2Me). ESI mass spec-
trometry (methanol) m/z 211.25 [C10H14N2O3+H]+, 233.05
2.2. Syntheses of ligands
Caution: some of the compounds prepared below are perchlorate
salts of organic-metal complexes and are potentially explosive. Even
small amounts of material should be handled with caution.
2,6-Bis(chloromethyl)-4-methylphenol,
[23]
2-(pyridyl-2-
ylmethylamino)ethanol [24], 2,20-(2-hydroxy-5-methyl-1,3-phe
nylene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethanol
(CH3H3L1) and the corresponding dizinc(II) complex [25] were
prepared as described previously.
[C10H14N2O3+Na]+ FT-IR spectroscopy ( , cmꢂ1) 3325.6 (m, O–H
v
str); 3117.9, 3083.4 (m, N–H sym/asym str); 2952.9 (w, C–H str);
1727.2 (s, C=O str); 1298.5, 1234.5 (s, CO2Me sym/asym def);
1063.6 (m, C–O str); 758.6 (m, py-H def).
2.2.1. Synthesis of methyl-6-(hydroxymethyl)picolinate
This compound was synthesized after a published procedure
[26]. Dimethyl pyridine-2,6-dicarboxylate (2.00 g, 10.2 mmol)
was dissolved in a 7:3 mixture of dry methanol/chloroform
(100 mL), then sodium borohydride (390 mg, 10.3 mmol) was
added in small portions at 0 °C. After stirring for 3 h at room tem-
perature, the solution was neutralized with saturated ammonium
chloride solution, concentrated to 30 mL in vacuo, and extracted
with dichloromethane (3 ꢁ 50 mL). The organic layers were com-
bined, dried over sodium sulfate, and the solvent was removed in
vacuo. The resulting oil was purified using column chromatography
(hexane/ethyl acetate 1:1) to yield 950 mg (55.9%) of a colorless oil
which crystallized upon standing. 1H NMR (CDCl3, 500.13 MHz); d
3.98 (s, 3H, OCH3); 4.85 (s, 2H, CH2OH); 7.52 (d, 1H, pyH,
J = 7.8 Hz); 7.85 (t, 1H, pyH, J = 7.8 Hz); 8.02 (d, 1H, pyH,
J = 7.7 Hz). 13C NMR (CDCl3, 100.62 MHz); d 52.9 (OCH3); 64.2
(CH2OH); 124.0 (pyCH); 124.5 (pyCH); 138.4 (pyCH); 146.4 (pyC);
160.2 (pyC); 165.5 (CO2CH3). ESI mass spectrometry (methanol)
2.2.4. Dimethyl-6,60-(2-hydroxy-5-methyl-1,3-phenylene)bis-
(methylene)bis((2-hydroxyethyl) azanediyl)bis(methylene)-
dipicolinate (Me2H3L4)
Methyl-6-(2-hydroxyethylamino)methyl picolinate (0.58 g,
2.7 mmol) was dissolved together with triethylamine (0.27 g) in tet-
rahydrofuran (2 mL), and 2,6-bis(dichloromethyl)cresol (0.28 g,
1.3 mmol) in dichloromethane (2 mL) was added dropwise at 0 °C.
The reaction was allowed to warm to room temperature, and was
stirred for 3 days. The yellow reaction mixture was filtered and con-
centrated in vacuo. The oily residue was taken up in dichlorometh-
ane (5 mL) and dried over sodium sulfate. After filtration and
removal of the solvent in vacuo, the desired ligand was obtained as
yellowish oil in 89% yield (650 mg). 1H NMR (CDCl3, 500.13 MHz)
d 2.15 (s, 3H, CH3); 2.75 (t, 4H, NCH2CH2, J = 6.1 Hz); 3.69 (t, 4H,
NCH2CH2, J = 6.0 Hz); 3.71 (s, 4H, arCH2N); 3.73 (s, 4H, pyCH2N);
3.85 (s, 6H, OCH3); 4.37 (s, 2H, OH); 6.99 (s, 2H, arH); 7.61 (dd, 2H,
pyH, J = 7.4, 1.4 Hz); 7.80 (m, 2H, pyH, J = 7.7, 1.4 Hz); 7.91 (m, 2H,
pyH, J = 7.6 Hz). 13C NMR (CDCl3, 100.62 MHz) d 20.2 (CH3); 52.4
(OCH3); 54.4 (arCH2N); 55.7 (NCH2CH2); 58.5 (NCH2CH2); 59.3
(pyCH2N); 123.2 (pyCH); 123.4 (arCCH2); 126.4 (pyCH); 126.7
(arCCH3); 129.2 (arCH); 137.8 (pyCH); 146.6 (pyCCO2CH3); 153.1
(arCOH); 159.6 (pyCCH2); 165.3 (CO2CH3). ESI mass spectrometry
m/z 168.24 [C8H9NO3+H]+. FT-IR spectroscopy ( , cmꢂ1) 3284.8 (s,
v
O–H str); 2956.1, 2908.8, 2849.3 (w, CH2 str); 1741.3 (s, C=O str);
1290.4 (s, C–O str); 1145.5 (s, C–O str); 759.4 (m, Py-H def). Melt-
ing point 85.8–90.6 °C lit. m.p. 88 °C [27].
2.2.2. Synthesis of methyl-6-formylpicolinate
This compound was synthesized after a modified published pro-
cedure [28]. Methyl-6-(hydroxymethyl)picolinate (1 g, 6 mmol)
was dissolved in dichloromethane (25 mL), and manganese dioxide
(5.2 g, 80 mmol) was added. The suspension was stirred at room
temperature for 5 days and subsequently filtered through Celite.
The filtrate was concentrated in vacuo, and the crude product
was purified using flash column chromatography (dichlorometh-
ane/methanol 85:15, I2 stain, Rf = 0.73) to yield 206 mg (21%) of a
white solid (250 mg of starting material were re-isolated). 1H
NMR (CDCl3, 500.13 MHz); d 4.05 (s, 3H, OCH3); 8.03 (td, 1H,
pyH, J = 7.7, 0.8 Hz); 8.12 (dd, 1H, pyH, J = 7.8, 1.2 Hz); 8.33 (dd,
1H, pyH, J = 7.7, 1.2 Hz); 10.17 (d, 1H, CHO, J = 0.8 Hz). 13C NMR
(CDCl3, 100.62 MHz); d 53.3 (OCH3); 124.4 (pyCH); 129.0 (pyCH);
138.4 (pyCH); 148.6 (pyC); 152.8 (pyC); 164.9 (CO2CH3); 192.6
(CHO). ESI mass spectrometry (methanol) m/z 188.30 [C8H7NO3+-
(methanol, neg) m/z 551.78 [C29H35N4O7]ꢂ. FT-IR spectroscopy (
v,
cmꢂ1) 3284.4 (b, O–H str); 2951.4 (m, C–H str); 1725.2 (m, C=O
str); 1226.4, 1138.6 (m, C–O str); 728.8 (s, py-H).
2.2.5. Synthesis of 2-((6-methylpyridin-2-yl)amino)ethanol
6-Methylpicolin-aldehyde (1.54 g, 0.013 mol) was dissolved in
24 mL of MeOH and cooled to 0 °C. 2-Aminoethanol (0.77 mL,
0.013 mol) was added dropwise at 0 °C, after which the reaction
was allowed to reach room temperature and then stirred for an
additional 2 h. After this time, thin layer chromatography (TLC)
analysis (100% MeOH) showed almost complete disappearance of
the starting material. The reaction was once again cooled to 0 °C
and, while monitoring by TLC, NaBH4 (0.30 g, 0.008 mol) was
added in small portions until disappearance of the intermediate
imine. Water (24 mL) was then added in small portions at 0 °C.
The volume of the solution was reduced to ꢃ24 mL by rotary evap-
oration. This solution was extracted with 3 ꢁ ꢃ20 mL DCM, the
combined organic layers were washed with 2 ꢁ 20 mL brine and
Na]+. FT-IR spectroscopy ( , cmꢂ1) 2860.8 (w, C–H str); 1719.4,
v
1708.2 (s, C=O str); 1140.9 (m, C–O str); 762.5 (m, py-H def).
m.p. 82.4–84.2 °C.

1338
K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
dried over anhydrous Na2SO4, and the dichloromethane was re-
moved by rotary evaporation. viscous amber oil resulted
Anal. calc. for C27H39ClN4O16Zn2: C, 38.5; H, 4.67; N, 6.65: Found: C,
38.9; H, 4.66; N, 6.66%. 1H NMR ((CD3)2SO) d: 1.97 (3H, s, ar-CH3),
2.80 (2H, br m, N–HCH–CH2–OH), 3.04 (2H, br m, N–HCH–CH2–
OH), 3.23 (2H, d, J = 11.3 Hz, ar/py-HCH–N), 3.66 (2H, br m, HCH-
OH), 3.72 (2H, d, J = 17.7, ar/py-HCH–N), 3.84 (2H, d, J = 11.5 Hz,
ar/py-HCH–N), 3.90 (2H, d, J = 17.7 Hz, ar/py-HCH–N), 4.00 (2H, br
m, HCH–OH), 6.65 (2H, s), 7.30 (2H, d, J = 7.7 Hz, py-H), 7.82 (2H,
d, J = 7.6 Hz, py-H), 7.94 (2H, t, J = 7.7 Hz, 40-py-H). 13C NMR
((CD3)2SO) d: 19.87 (ar-CH3), 54.47 (CH2), 56.76 (CH2), 56.88
(CH2), 57.44 (CH2-OH), 121.08 (py-CH), 123.92 (py-CH2), 125.00
(ar-C), 125.83 (ar-C), 131.46 (ar-CH), 141.33 (40-py-CH), 148.29
(py-C), 153.65 (py-C), 158.79 (ar-C–OH), 165.04 (CO2ꢂ). ESI mass
spectrometry (CH3OH): m/z 649.1 (70%); 651.1 (99%); 653.1
(100%) 653.02. (Calculated for [C27H29N4O7Zn2]+ 649.1 (82%);
651.1 (99%); 653.1 (100%)). Crystals suitable for X-ray structure
determination, but which desiccated readily on removal from sol-
vent, were grown on prolonged standing of the reaction mixture
in an ethanol/methanol solvent mixture. These crystals were subse-
quently characterized crystallographically as [Zn2(H2L4)(H2O)2](-
ClO4) 2CH3OHꢀ1.5H2OꢀCH3CH2OH.
A
(0.94 g, 44.5% yield). 1H NMR (400 MHz, CDCl3) d: 2.52 (3H, s, py-
CH3), 2.85 (2H, t, J = 5.1 Hz, N–CH2–CH2–OH), 3.63 (2H, t,
J = 5.1 Hz, CH2–OH), 3.89 (2H, s, py-CH2–N), 7.01 (1H, d, J = 7.6
Hz, py-H), 7.04 (1H, d, J = 7.6 Hz, py-H), 7.51 (1H, t, J = 7.6 Hz, 40-
py-H). 13C NMR (100 MHz, CDCl3) d: 24.33 (ar-CH3), 51.14 (CH2),
54.29 (CH2), 60.92 (CH2–OH), 119.15 (py-CH), 121.62 (py-CH),
136.86 (py-CH), 158.03 (py-C), 159.10 (py-C). ESI mass spectrome-
try: m/z = 167.1179 (calculated for [C9H14N2O+H]+ = 167.1179).
2.2.6. Synthesis of 2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(methy-
lene)bis(((6-methylpyridin-2-yl)methyl)azanediyl)diethanol (H3L5)
Triethylamine (0.78 mL, 6 mmol) was added dropwise to a solu-
tion of 2-((6-methylpyridin-2-yl)amino)ethanol (0.94 g, 5.6 mmol)
in 20 mL of dry tetrahydrofuran at 0 °C. To this was added a solution
of 2,6-bis(chloromethyl)-p-cresol (0.57 g, 2.8 mmol) in 20 mL of
dichloromethane at 0 °C. The resulting pale yellow solution was al-
lowed to reach room temperature and then stirred overnight. The
mixture was filtered to remove the white precipitate of triethyl-
amine hydrochloride, and the solvent was removed under vacuum,
yielding the product as a pale yellow wax (1.28 g, 99.1% yield). 1H
NMR (500 MHz, CD3OD) d: 2.18 (3H, s, py-CH3), 2.50 (6H, s, py-
CH3), 2.68 (4H, t, J = 5.0 Hz, N–CH2–CH2–OH), 3.66 (4H, t, J = 5.0 Hz,
CH2–OH), 3.70 (4H, s, ar-CH2–N), 3.77 (4H, s, py-CH2–N), 6.86 (2H,
s, ar-H), 7.10 (2H, d, J = 7.7 Hz, 50-py-H), 7.25 (2H, d, J = 7.7 Hz, 30-
py-H), 7.60 (2H, t, J = 7.7 Hz, 40-py-H). 13C NMR (125 MHz, CD3OD,
328K) d: 20.49 (ar-CH3), 23.82 (py-CH3), 56.64 (ar-CH2–N), 57.34
(N–CH2–CH2–OH), 60.56 (N–CH2–CH2–OH), 60.87 (py-CH2–N),
121.79 (30-py–CH), 123.04 (50-py-CH), 124.95 (ar-C–CH2), 129.04
(ar-C–CH3), 131.01 (ar-CH), 138.56 (40-py-CH), 154.72 (ar-C–OH),
158.86 (60-py-C–CH3), 159.69 (20-py-C–CH2). ESI mass spectrome-
try: m/z = 465.2872 (calculated for [C27H36N4O3+H]+ = 465.2860).
2.3.2. Synthesis of [Zn2(H2L5)(CH3CO2)(H2O)](PF6)2ꢀ2H2O
H3L5 (0.50 g, 0.001 mol) was dissolved in 30 mL of MeOH, to
which was added zinc acetate dihydrate (0.60 g, 3 mmol). The
resulting pale yellow solution was then refluxed for 0.5 h. After this
time, the total solvent volume was reduced to ꢃ15 mL by rotary
evaporation and to the solution was added NaPF6 (0.53 g,
0.003 mol). The solution was heated to ꢃ60 °C in a water bath
for ꢃ10 min, gravity filtered, and stored at 0 °C. Colorless crystals
(0.52 g, 52% yield), which desiccated in open air, were collected.
All attempts to produce diffraction quality crystals were unsuc-
cessful. Elemental Anal. Calc. for C29H44F12N4O8P2Zn2: C, 34.92; H,
4.45; N, 5.62; Zn, 13.12. Found: C, 34.87; H, 4.25; N, 5.55; Zn,
12.97%. 1H NMR (500 MHz, (CD3OD) d: 2.03 (3H, s, ar-CH3), 2.16
(3H, s, CH3–CO2ꢂ), 2.77 (6H, s, py-CH3), 2.99 (2H, dt, J = 12.3 Hz,
3.4 Hz, CH2), 3.08–3.23 (4H, br m, CH2), 3.77–3.86 (6H, s overlap-
ping with br m, CH2), 3.98 (2H, br m, CH2), 4.14 (2H, d,
J = 12.0 Hz, CH2), 6.64 (2H, s, ar-CH), 6.80 (2H, d, J = 6.8 Hz, 30-py-
H), 7.19 (2H, d, J = 7.7 Hz, 50-py-H), 7.58 (2H, t, J = 7.7 Hz, 40-py-
H). 13C NMR (100 MHz, (CD3)2SO) d: 19.93 (ar-CH3), 23.80
(CH3CO2ꢂ), 23.99 (CH3-py), 56.79 (CH2), 58.65 (2xCH2), 59.57
(CH2), 121.27 (30-py-CH), 124.06 (ar-C), 125.43 (50-ar-CH), 128.29
(ar-C), 133.64 (ar-CH), 141.38 (40-py-CH), 156.21 (py-C), 159.17
(ar-C-OH, py-C), 182.00 (CH3CO2ꢂ). ESI mass spectrometry:
m/z = 589.08 (calculated for [C27H33N4O3Zn2]+ = 589.11).
2.2.7. Synthesis of 6,60-(2-hydroxy-5-methyl-1,3-phenylene)bis
(methylene)bis((2-hydroxyethyl)azanediyl) bis(methylene)dipicolina-
mide (H3L6)
The synthesis was adapted from a previously published proce-
dure [29]. Me2H3L4 (1.04 g, 1 mmol) was suspended in ꢃ15 mL
of concentrated NH4OH and stirred overnight in a sealed reaction
vessel, resulting in the formation of a yellow waxy solid. The sus-
pension was sonicated for four hours, then the solvent was re-
moved, affording the product as a yellow wax (0.97 g, 98.6%
yield). 1H NMR (400 MHz, CDCl3) d: 2.17 (3H, s, ar-CH3), 2.75
(4H, t, J = 5.3 Hz, N–CH2–CH2–OH), 3.66 (4H, t, J = 5.3 Hz, N–CH2–
CH2–OH), 3.75 (4H, s, ar-CH2–N), 3.88 (4H, s, py-CH2–N), 6.78
(2H, s, ar-H), 7.37 (2H, dd, J = 7.7 Hz, 0.9 Hz, py-H), 7.74 (2H, t,
J = 7.7 Hz, 40-py-H), 8.03 (2H, dd, J = 7.7 Hz, 0.9 Hz, py-H), 8.22
(2H, broad, NH2). 13C NMR (100 MHz, CDCl3) d: 20.39 (ar-CH3),
55.85 (CH2), 56.24 (CH2), 59.26 (CH2), 59.46 (CH2), 121.10 (ar/py-
C), 123.31 (ar/py-C), 125.58 (ar/py-C), 128.10 (ar/py-C), 130.21
(ar/py-C), 137.73 (ar/py-C), 149.43 (ar/py-C), 153.57 (ar/py-C),
157.56 (ar/py-C), 166.82 (CONH2). ESI mass spectrometry: m/
z = 523.26 (calculated for [C27H34N6O5+H]+ = 523.27), m/z = 545.25
(calculated for [C27H34N6O5+Na]+ = 545.25).
2.3.3. Attempted synthesis of [Zn2(H2L6)(H2O)2](BF4)
H3L6 (0.97 g, 2 mmol) was dissolved in 60 mL of MeOH, to
which was added zinc acetate dihydrate (0.70 g, 4 mmol). The
resulting pale yellow solution was then refluxed for 0.5 h. After this
time, the total solvent volume was reduced to ꢃ15 mL by rotary
evaporation, and NaBF4 was added, resulting in colorless product
in low yield. ESI mass spectrometry and microanalysis indicated
that the complex was [Zn2(H2L4)(H2O)2](BF4)ꢀ3H2O. Elemental
Anal. calc. for C27H35BF4N4O10Zn2: C, 40.88; H, 4.45; N, 7.06. Found:
C, 40.43; H, 4.24; N, 6.97%. LRMS (CH3OH): m/z 649.1 (70%); 651.1
(99%); 653.1 (100%) 653.02. (Calculated for [C27H29N4O7Zn2]+ 649.1
(82%); 651.1 (99%); 653.1 (100%)).
2.3. Synthesis of metal complexes
2.3.1. Synthesis of [Zn2(H2L4)(H2O)2](ClO4)ꢀ3H2O
Me2H3L4 (0.35 g, 0.6 mmol) was dissolved in MeOH (20 mL), to
which was added zinc acetate dihydrate (0.60 g, 1.3 mmol). The
resulting pale yellow solution was refluxed for 0.5 h. The total sol-
vent volume was reduced to ꢃ6 mL, Bu4NClO4 (0.33 g, 1 mmol) was
added, and the mixture was heated to ꢃ60 °C in a water bath for
ꢃ10 min and then allowed to concentrate by slow evaporation at
room temperature, resulting in a colorless solid (0.060 g, 12% yield).
2.4. Crystallography
X-ray diffraction data for a crystal of [Zn2(H2L4)(H2O)2](ClO4)ꢀ
2CH3OHꢀ1.5H2OꢀCH3CH2OH were collected on a Bruker APEX II
CCD diffractometer, with graphite monochromated Mo
Ka
(k = 0.71069 Å) radiation at 90 K, in a nitrogen gas stream. Intensi-
ties were corrected for Lorentz polarization effects, and a multiscan

K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
1339
Table 1
in 10 mm quartz cuvettes. The initial rate method was employed
and assays were measured such that the initial linear portion of
the data was used for analysis. Product formation was determined
at 25 °C by monitoring the formation of 2,4-dinitrophenol. The
extinction coefficient for 2,4-dinitrophenolate at 400 nm, through-
out the pH range studied, is 12,100 Mꢂ1 cmꢂ1 [32,33]. For each as-
say, corrections for the rate of autohydrolysis were applied. An
aqueous multi-component buffer (50 mM in each of 2-(N-morpho-
lino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES) and 2-(N-cyclohexylamino)ethane
sulfonic acid (CHES), with the ionic strength controlled by 250 mM
LiClO4) was used. Assays were carried out in 50:50 MeCN:buffer,
with substrate and complex initially dissolved in MeCN. Assays con-
Summary of crystallographic data for [Zn2(H2L4)(H2O)2](ClO4)ꢀ2CH3OHꢀ1.5H2Oꢀ
CH3CH2OH.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C31 H47 Cl N4 O17.50 Zn2
921.92
90(2) K
0.71069 Å
Triclinic
ꢀ
P1
Unit cell dimensions
a (Å)
b (Å)
c (Å)
12.703(3)
12.852(2)
13.240(3)
75.919(5)
69.716(9)
68.503(9)
1869.7(7)
a
(°)
b (°)
ducted to investigate pH dependence were 40 lM in complex and
c
(°)
V (Å3)
5 mM in BDNPP and showed no significant buffer effects. Assays
used to assess the substrate and complex dependence were carried
out at pH 10.5 for the complex. [Substrate] dependence assays were
Z
Dcalc
2
1.638 Mg/m3
16106
Reflections collected
Independent reflections
Refinement method
Goodness-of-fit (GOF) on F2
Final R indices [I > 2
R indices (all data)
40
l
M in complex and 1–9 mM in BDNPP, and [complex] depen-
M in complex and 5 mM in BDNPP.
6909 [R(int) = 0.0170]
Full-matrix least-squares on F2
1.048
R1 = 0.0334, wR2 = 0.0910
R1 = 0.0363, wR2 = 0.0932
dence assays were 20–160
l
The change in absorbance produced by hydrolysis of BDNPP by free
Zn(II) (from Zn(ClO4)2), in the same concentration as the complex,
did not differ significantly from autohydrolysis values. All data were
fitted by non-linear least squares regression analysis [34].
r(I)]
3. Results and discussion
Table 2
Selected bond lengths (Å) and angles (°) for [Zn2(H2L4)(H2O)2](ClO4)ꢀ2CH3OHꢀ
3.1. Synthesis
1.5H2OꢀCH3CH2OH.
Zn(1)–O(1)
Zn(1)–O(8)
Zn(1)–N(3)
Zn(2)–O(1)
Zn(2)–O(7)
Zn(2)–N(1)
Zn(1)...Zn(2)
2.0597(17)
2.1448(18)
2.168(2)
2.0405(18)
2.1087(18)
2.194(2)
3.60
Zn(1)–O(5)
Zn(1)–O(10)
Zn(1)–N(4)
Zn(2)–O(4)
Zn(2)–O(11)
Zn(2)–N(2)
2.3528(19)
1.9808(19)
2.053(2)
2.287(2)
2.015(2)
The ligand dimethyl-6,60-(2-hydroxy-5-methyl-1,3-phenylene)
bis(methylene)bis((2-hydroxyethyl)azanediyl)bis(methylene)di-pi
colinate (H3Me2L4) has been synthesized through a substitution
reaction between 2,6-bis(chloromethyl)-4-methylphenol and
methyl-6-((2-hydroxyethylamino)methyl)picolinate; the ligand
was isolated as the methyl ester. Methyl 6-formylpicolinate was
prepared by reduction of dimethyl pyridine-2,6-dicarboxylate to
the corresponding mono-alcohol, and then reaction with MnO2
to prepare the aldehyde. H3L5 was prepared by the reaction of 2-
((6-methylpyridin-2-yl)amino)ethanol with 2,6-bis(chloromethyl)-
4-methylphenol. The amide ligand 6,60-(2-hydroxy-5-methyl-1,
3-phenylene)bis(methylene)bis((2-hydroxyethyl)azanediyl)bis
(methylene)dipicolinamide (H3L6) was prepared by the reaction of
H3Me2L4 with aqueous ammonia. The reaction scheme employed
is shown in Scheme 1. The complexes were prepared after reaction
with zinc(II) acetate; in both cases no attempts were made to opti-
mize yields, and the complexes proved to be difficult to obtain as
X-ray quality crystals. Reaction of the methyl ester H3Me2L4 with
the metal salt results in hydrolysis of the ester, an effect observed
previously with similar ligands [35]. Crystals of [Zn2(H2L4)(H2O)2](-
ClO4) were obtained in low yield, although they contained mixed
solvent molecules in the lattice, and they desiccated on removal
from the parent solvent. The kinetic studies reported herein em-
ployed the crystallized sample. On reaction of H3L6 with zinc(II) ace-
tate hydrolysis of the amide occurred resulting in the isolation of the
2.058(2)
Zn(1)–O(1)–Zn(2)
N(3)–Zn(1)–N(4)
N(1)–Zn(2)–N(2)
122.94(8)
78.68(8)
77.93(8)
O(1)–Zn(1)–N(3)
O(1)–Zn(2)–N(1)
93.22(8)
93.48(7)
absorption correction was applied. The structure was solved by di-
rect methods using SIR97 and refined on F2 using SHELXL97 run-
ning within the WinGX interface [30]. Plots were drawn using
ORTEP3 [31]. All non-hydrogen atoms were refined with aniso-
tropic thermal parameters. The crystal structure contained disor-
ꢂ
dered components. The ClO4 counter ion showed translational
disorder over two sites, with site occupancy factors of 0.69 and
0.31. Both of the methanol solvent molecules showed translational
disorder over two sites, with site occupancy factors of 0.70 and
0.30 for C33 and O15, and 0.57 and 0.43 for C32 and O16. Four
H2O molecules were found to only partially occupy sites, with
two molecules having site occupancy factors of 0.50, and two of
0.25. Selected crystal data and some details of refinements are gi-
ven in Table 1. Selected bond distances and angles are presented in
Table 2. X-ray data were deposited with the Cambridge Crystallo-
graphic Data Center CCDC-793400. These data can be obtained free
of charge from the Cambridge Crystallographic Data Center via
http://www.ccdc.cam.ac.uk/data_request/cif.
dizinc(II) complex of H2L43ꢂ
.
The nomenclature employed for the ligands follows that de-
scribed previously and denotes the number of removable protons
upon complexation (Chart 1) [36]. The free acid ligand, 6,60-(2-hy-
droxy-5-methyl-1,3-phenylene)bis(methylene)bis((2-hydroxyethyl)
azanediyl)bis(methylene)dipicolinic acid was not isolated, hence
the nomenclature Me2H3L4 denotes the methyl ester and poten-
tially three sites for deprotonation, the phenyl and the two pendant
alcohols. Upon reaction with zinc acetate and after hydrolysis of the
methyl ester groups, the resulting complex is designated as
[Zn2(H2L4)(H2O)2](ClO4) (H2L43ꢂ), with two carboxylate donors
and the phenoxide deprotonated; the two pendant alcohols are
2.5. Catalytic studies
The phosphoesterase-like activity of the complexes was deter-
mined by measuring hydrolysis of the substrate BDNPP. A Varian
Cary50 Bio UV/Visible spectrophotometer with a Peltier tempera-
ture controller was used to measure changes in absorbance values

1340
K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
Scheme 1. Synthesis of 2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene) bis((pyridin-2-ylmethyl)azanediyl)diethanol (CH3H3L1), dimethyl-6,60-(2-hydroxy-5-
methyl-1,3-phenylene)bis(methylene)bis((2-hydroxyethyl) azanediyl)bis(methylene) dipicolinate (Me2H3L4), 2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis
(((6-methylpyridin-2-yl)methyl) azanediyl) diethanol (H3L5) and 6,60-(2-hydroxy-5-methyl-1,3-phenylene)bis (methylene)bis((2-hydroxyethyl)azanediyl) bis (methy-
lene)dipicolinamide (H3L6).
graphic data are shown in Table 1, selected bond lengths and an-
gles are displayed in Table 2. An ORTEP [31] plot of the complex
cation is shown in Fig. 1. For [Zn2(H2L4)(H2O)2](ClO4)ꢀ2CH3OHꢀ
1.5H2OꢀCH3CH2OH, the ligand has the two carboxylic acid groups
and the bridging phenol moiety deprotonated; the pendant alcohol
moieties are protonated. Both of the zinc(II) centers display a dis-
torted six coordinate geometry. In both cases the N2O4 coordina-
tion environment of the metal ions is composed of the tertiary
amine (Zn(1)–N(3), 2.168(2) Å; Zn(2)–N(1), 2.194(2) Å), the pyri-
dine nitrogen (Zn(1)–N(4), 2.053(2) Å; Zn(2)–N(2), 2.058(2) Å),
the alcohol (Zn(1)–O(5), 2.3528(19) Å; Zn(2)–O(4), 2.287(2) Å),
the deprotonated carboxylate (Zn(1)–O(8), 2.1448(18) Å; Zn(2)–
O(7), 2.1087(18) Å), and
a water molecule (Zn(1)–O(10),
1.9808(19) Å; Zn(2)–O(11), 2.015(2) Å). The six coordinate geome-
try is completed by the bridging oxygen from the phenoxide
(Zn(1)–O(1), 2.0597(17) Å; Zn(2)–O(1), 2.0405(18) Å) with Zn(1)–
O(1)–Zn(2), 122.94(8)°; the Zn(1)–Zn(2) distance is 3.608 Å. The
Chart 1. Ligand abbreviations. CH3H3L1 = 2,20-(2-hydroxy-5-methyl-1,3-pheny-
lene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethanol;[25] CH3HL2 = 2,
6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-4-methylphenol;
BrHL2 = 4-bromo-2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)
phenol; NO2HL2 = 2,6-bis(((2-methoxyethyl)(pyridin-2-ylmethyl)amino)methyl)-
4-nitrophenol; CH3HL3 = 4-methyl-2,6-bis(((2-phenoxyethyl)(pyridin-2-ylmethyl)
amino)methyl)phenol [36]; H5L4 = 6,60-((((2-hydroxy-5-methyl-1,3-phenylene)bis
(methylene))bis((2-hydroxyethyl)azanediyl))bis(methylene)) dipicolinic acid; H3L5 =
2,20-(2-hydroxy-5-methyl-1,3-phenylene)bis(methylene)bis(((6-methylpyridin-2-yl)
methyl)azanediyl) diethanol; (H3L6) = 6,60-(2-hydroxy-5-methyl-1,3-phenylene)
bis(methylene) bis((2-hydroxyethyl)azanediyl) bis (methylene)dipicolinamide.
protonated. Similarly, for H3L5 the ligand has potentially three sites
for deprotonation, the phenyl and the two pendant alcohols; char-
acterization of the complex as [Zn2(H2L5)(CH3CO2)(H2O)](PF6)2ꢀ
2H2O implies that the phenol is deprotonated.
3.2. Discussion of X-ray structure
The
complex
[Zn2(H2L4)(H2O)2](ClO4)ꢀ2CH3OHꢀ1.5H2Oꢀ
ꢀ
Fig. 1. ORTEP [31] plot of [Zn2(H2L4)(H2O)2](ClO4)ꢀ2CH3OHꢀ1.5H2OꢀCH3CH2OH with
50% probability level of thermal ellipsoids. Hydrogen atoms, counter ions and non-
coordinated solvent molecules have been omitted for clarity, as have carbon atom
labels.
CH3CH2OH crystallized in the triclinic P1 space group. The struc-
ture is composed of the complex cation, a perchlorate anion and
solvent molecules completing the structure. Selected crystallo-

K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
1341
1.6e-8
Zn–O and Zn–N distances appear typical of those reported for the
di-zinc(II) complex of 2,20-(2-hydroxy-5-methyl-1,3-pheny-
lene)bis(methylene)bis((pyridin-2-ylmethyl)azanediyl)diethanol
(CH3H3L1; Chart 1) [25], particularly the alcohol Zn–O distances
(2.3528(19), 2.287(2) Å) which appear typical for a protonated
alcohol bound to zinc(II) [25].
1.4e-8
1.2e-8
1.0e-8
8.0e-9
6.0e-9
4.0e-9
2.0e-9
0.0
3.3. Mass spectrometry
Mass spectral analysis, determined with solvent conditions em-
ployed in the hydrolytic studies, H2O:CH3CN (1:1), shows that the
complexes exist as dinuclear zinc species with the isotopic pat-
terns for Zn2 species being distinctly different from that observed
for a mono-zinc species. Thus, species indicative of [Zn2(H2L4)]+
(m/z 649.1 (82.5%; 651.1 (99.2%); 653.1 (100%)) (Fig. 2) and
[Zn2(L5)]+ (m/z 589.1 (83.6%); 591.1 (99.3%); 593.1 (100%)) were
observed. The mass spectral evidence suggests that under the sol-
vent conditions employed in the kinetic studies the acetate ligands
are lost [36].
5
6
7
8
9
10
11
pH
2.5e-8
2.0e-8
1.5e-8
1.0e-8
5.0e-9
0.0
3.4. Phosphodiesterase-like activity
Phosphatase-like activity was measured at various pH values
ranging from 5 to 10.5 using the activated substrate BDNPP
(Fig. 3(a)). For both complexes the data were consistent with the
presence of one protonation equilibrium and were fitted to an
equation derived for a monoprotic system (Eq. (1)) [37].
Vmax
ꢀ
ꢁ
V0
¼
ð1Þ
Hþ
½
ꢄ
1 þ
Kes
0
2
4
6
8
10
[BDNPP] (mM)
Fig. 3. pH dependence and substrate concentration dependence of rate of BDNPP
cleavage by [Zn2(H2L4)(H2O)2](ClO4) (o) and [Zn2(H2L5)(CH3CO2)(H2O)](PF6)2 ) at
(D
25 °C; aqueous multi-component buffer (50 mM each of MES, HEPES and CHES),
ionic strength controlled by 250 mM LiClO4; 50:50 MeCN:buffer).
Here, V0 is the initial reaction rate, Vmax is the maximum rate with
BDNPP as substrate (5 mM), and Kes (=Ka) is the protonation equilib-
rium constant relevant to catalysis. The catalytically relevant
species derived from [Zn2(H2L4)(H2O)2]+ and [Zn2(H2L5) (CH3CO2)
(H2O)]2+ exhibited pKas of 9.4 0.05 and 6.6 0.05, respectively.
The substrate concentration dependence of catalysis was also
measured at pH 10.5 and 8.0 for [Zn2(H2L4)(H2O)2]+ and
[Zn2(H2L5)(CH3CO2)(H2O)]2+, respectively. Michaelis–Menten type
behavior was observed (Fig. 3(b)) and the data fitted using non-lin-
ear least square analysis (Eq. (2)) gave the Michaelis constants, Vmax
and kcat (Vmax/[complex]) [37].
Vmax½Sꢄ
V0
¼
ð2Þ
Km þ ½Sꢄ
Here, Km is the Michaelis constant and [S] the substrate concen-
tration. [Zn2(H2L4)(H2O)2]+ and [Zn2(H2L5)(CH3CO2)(H2O)]2+
exhibited kcat = 3.52 0.03 ꢁ 10ꢂ4 and 1.27 0.04 ꢁ 10ꢂ3 sꢂ1
(Km = 6.7 0.9; 13.8 1.5 mM), respectively. The rate of hydrolysis
of BDNPP (5 mM) was linear for complex concentrations from 20 to
160 lM.
3.5. Mechanism of reaction
Data for the rate of hydrolysis of BDNPP with a range of compa-
rable complexes are shown in Table 3. The effect of electron with-
Fig. 2. ES-mass spectrum of the complex [Zn2(H2L4)]+ recorded in H2O:CH3CN
(1:1); inset (a) the isotopic pattern for the envelope at m/z 651.1; (b) calculated
isotopic pattern indicative of a dizinc(II) complex.
drawing groups para- to
a phenolic oxygen donor on the

1342
K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
Table 3
Data for the rate of hydrolysis of BDNPP with dizinc(II) complexes. IPCPMP = 2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(carboxylmethyl)-N-((2-pyri-
dyl)methyl)amino methyl)-4-methylphenol [56].
Complex
kcat (s-1
)
Km (mM)
kcat/Km (Mꢂ1 sꢂ1
)
pKa
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6)
[Zn2(CH3L2)(CH3COO)2](PF6) [36]
[Zn2(BrL2)(CH3COO)2](PF6) [36]
[Zn2(NO2L2)(CH3COO)2](PF6) [36]
[Zn2(CH3L3)(CH3COO)2](PF6) [36]
[(Zn2(IPCPMP)(CH3COO))2](PF6)2 [56]
[Zn2(H2L4)(H2O)2](ClO4)
1.07 0.04 ꢁ 10ꢂ3
5.70 0.04 ꢁ 10ꢂ3
1.90 0.04 ꢁ 10ꢂ3
0.76 0.04 ꢁ 10ꢂ3
3.60 0.04 ꢁ 10ꢂ3
6.4 ꢁ 10ꢂ4
12.5 7.0
20.8 5.0
7.1 4.0
6.5 1.4
18.9 3.5
0.086
0.274
0.268
0.117
0.190
0.040
0.053
0.092
6.6
6.7
6.5
6.5
7.7
6.6
9.4
6.6
16
5
3.52 0.03 ꢁ 10ꢂ4
1.27 0.04 ꢁ 10ꢂ3
6.7 0.9
13.8 1.5
[Zn2(H2L5)(CH3CO2)(H2O)](PF6)2
magnitude of kcat has shown that, generally, the more electron-
withdrawing the substituent the lower the kcat value [36,38]. For
the complexes [Zn2(H2L4)(H2O)2]+ and [Zn2(H2L5)(CH3CO2)(-
H2O)]2+ and [Zn2(CH3HL1)(CH3CO2)(H2O)]+ with COOꢂ, CH3 and H
substituents meta- to the pyridine nitrogen, a Hammett style plot
shows a linear relationship for the inductive influence of the sub-
As observed previously, the trend is less well defined for the
respective pKa values [36]. What is evident is the difference in the
kinetically relevant pKa between [Zn2(H2L4)(H2O)2](ClO4) (pKa
9.4) and that exhibited by [Zn2(L5)]+ (pKa 6.6) and other similar
dizinc(II) complexes (Table 3). Complexes with -L1, -L4 and -L5
type ligands (Table 3) possess two potentially nucleophilic entities
for the hydrolysis of the substrate BDNPP, Zn–OH and Zn–OR
(alkoxide), whereas those with -L2 type ligands (Chart 1) have the
alkoxide replaced by an ether moiety and the nucleophile is a Zn–
OH species [36]. Theoretical studies suggest that a zinc(II)-coordi-
nated alcohol has a lower pKa than a zinc(II)-coordinated water
[42] and is more reactive [42–46]. The pKa value of 9.4, determined
for the catalytically relevant pKa for [Zn2(H2L4)]+, is at the high end
of the range observed for terminally bound H2O ligands
[25,36,42,45–56], but not typical for a zinc(II) bound alcohol. The
pKa is similar to those reported for the aminopeptidase mimics
[Zn2(bocp)(CH3COO)2]BPh4 and [Zn2(bomp)(CH3COO)2]BPh4 (bocp =
4-chloro-2,6-bis(((2-methoxyethyl)(methoxymethyl)amino)methyl)
phenol; bomp = 2,6-bis(((2-methoxyethyl) (methoxymethyl)amino)
methyl)-4-methylphenol, (9.1 and 9.4, respectively) [57]. As well,
substitution of a neutral donor with an anionic ligand in the pri-
mary coordination sphere reduces the Lewis acidity of the zinc(II)
center, raising the pKa of the Zn–OH2 [51,58,59]. For example, the
zinc(II) complexes of the ligand 2-(bis(pyridin-2-ylmethyl)
amino)acetic acid, and similar ligands, display a pKas typically
around 9 [58,60–63]. In addition, the higher coordination number
of the Zn(II) ions will also contribute to the higher pKa [64].
stituent on both the rate of the BDNPP hydrolysis reaction (kcat
)
and with Km (Fig. 4) [39,40]. The sequence of kcat with m-
CH3 > m-H > m-COOꢂ is in accord with previous studies with Fe(III)
complexes, and is ascribed to the Lewis acidity of the metal center
being directly affected by the donor/acceptor properties of the sub-
stituent [38]. The negative value of Hammett
q parameter for the
kcat plot (ꢂ1.3) reflects both the distance between the substituent
and the nucleophile, and the decreasing charge on the aromatic
ring [38,41].
-2.8
CH
3
-2.9
-3.0
-3.1
-3.2
-3.3
-3.4
-3.5
H
Comparison of the efficiency (kcat/Km) for [Zn2(H2L4)(H2O)2]+
and [Zn2(H2L5)(CH3CO2)(H2O)]2+, 0.053 Mꢂ1 sꢂ1 and 0.092 Mꢂ1 sꢂ1
,
COOH
respectively (Table 3), shows that the latter is similar to that for
[Zn2(CH3HL1)(CH3COO)2](PF6) and within the range reported for
the hydrolysis reactions of BDNPP for a number of heterodinuclear
complexes [32,56,65–68]. [Zn2(H2L4)(H2O)2]+ displays a lower kcat
and binds the substrate relatively more strongly than, for example,
[(Zn2(IPCPMP)(CH3COO))2](PF6)2 which displays a kcat of similar
magnitude and binds the substrate more weakly (kcat/Km = 0.040
Mꢂ1 sꢂ1) [56].
-0.1
0.0
0.1
0.2
0.3
0.4
σ
m
1.20
CH3
1.15
1.10
1.05
1.00
0.95
0.90
0.85
0.80
H
Structural
and
kinetic
similarities
between
[Zn2(CH3HL1)(CH3COO)(H2O)](PF6) [25] and [Zn2(H2L5)(CH3CO2)
(H2O)](PF6)2, coupled with the results of previous studies [36], sug-
gest that the active nucleophile for these complexes is a Zn(II)–OH
species. Structurally the situation for [Zn2(H2L4)(H2O)2]+ is differ-
ent. This complex has one exchangeable binding site on each of
the metal ions and, consequently, a more crowded active site.
Mechanistic proposals based on monodentate coordination of the
substrate include the terminal hydroxide on the second zinc(II) site
acting as the nucleophile, or acting as a general base, to deproto-
nate a neighboring water molecule which then attacks the phos-
phate ester [36,56,69,70]. If, however, the substrate BDNPP binds
in a bi-dentate manner, as suggested for bis(p-nitrophenyl)phos-
phate (BNPP) [25,71], then proposals for the hydrolysis could in-
clude (i) an additional binding site for a potentially nucleophilic
aqua/hydroxo moiety, necessitating a seven coordinate zinc(II) site,
COOH
-0.1
0.0
0.1
0.2
0.3
0.4
σ
m
Fig. 4. Linear correlation for the Hammett parameter (
log10(Km) ( ) for [Zn2(CH3HL1)(CH3COO)2](PF6) (m-H), [Zn2(H2L4)(H2O)2](ClO4) (m-
COOꢂ) and [Zn2(H2L5)(CH3CO2)(H2O)](PF6)2 (m-CH3).
rm) and (a) log10(kcat)-(o) (b)
D

K.E. Dalle et al. / Polyhedron 52 (2013) 1336–1343
1343
[25] J.W. Chen, X.Y. Wang, Y.G. Zhu, J. Lin, X.L. Yang, Y.Z. Li, Y. Lu, Z.J. Guo, Inorg.
Chem. 44 (2005) 3422.
[26] X. Zeng, D. Coquiere, A. Alenda, E. Garrier, T. Prange, Y. Li, O. Reinaud, I. Jabin,
Chem. Eur. J. 12 (2006) 6393.
[27] W. Mathes, W. Sauermilch, T. Klein, Chem. Berichte 86 (1953) 584.
[28] D.A. DeGoey, D.J. Grampovnik, C.A. Flentge, W.J. Flosi, H.-J. Chen, C.M. Yeung,
J.T. Randolph, L.L. Klein, T. Dekhtyar, L. Colletti, K.C. Marsh, V. Stoll, M. Mamo,
D.C. Morfitt, B. Nguyen, J.M. Schmidt, S.J. Swanson, H. Mo, W.M. Kati, A. Molla,
D.J. Kempf, J. Med. Chem. 52 (2009) 2571.
(ii) exchange of the coordinated alcohol for a potentially nucleo-
philic water molecule, (iii) the coordinated alcohol itself is the ac-
tive nucleophile, or (iv) the coordinated alcohol acts as a general
base to activate a neighboring water molecule. The enhanced coor-
dination environment appears unlikely, and previous studies have
questioned the mechanistic steps required for regeneration of the
active site if the alkoxide is the primary nucleophile [36]. Mecha-
nistic pathways involving monodentate coordination of the BDNPP
followed by attack of the nucleophile coordinated to the second
Zn(II) site [56], or a general base acting on a bidentate substrate,
appear possible [36,69,70]. The kinetically relevant pKa (9.4) asso-
ciated with the electronic and steric influences on the zinc(II) site
suggest that the former pathway is more probable.
[29] Y. Morisawa, M. Kataoka, T. Sakamoto, H. Nagahori, N. Kitano, K.I. Kusano, J.
Med. Chem. 21 (1978) 194.
[30] L.J. Farrugia, J. Appl. Cryst. 32 (1999) 837.
[31] L.J. Farrugia, J. Appl. Cryst. 30 (1997) 565.
[32] S.C. Batista, A. Neves, A.J. Bortoluzzi, I. Vencato, R.A. Peralta, B. Szpoganicz,
V.V.E. Aires, H. Terenzi, P.C. Severino, Inorg. Chem. Commun. 6 (2003) 1161.
[33] S.J. Smith, A. Casellato, K.S. Hadler, N. Mitic, M.J. Riley, A.J. Bortoluzzi, B.
Szpoganicz, G. Schenk, A. Neves, L.R. Gahan, J. Biol. Inorg. Chem. 12 (2007)
1207.
[34] SigmaPlot for Windows, Version 10, Systat Software Inc., San Jose, CA.
[35] A.K. Boudalis, R.E. Aston, S.J. Smith, R.E. Mirams, M.J. Riley, G. Schenk, A.G.
Blackman, L.R. Hanton, L.R. Gahan, Dalton Trans. (2007) 5132.
[36] L.J. Daumann, K.E. Dalle, G. Schenk, R.P. McGeary, P.V. Bernhardt, D.L. Ollis, L.R.
Gahan, Dalton Trans. 41 (2012) 1695.
[37] I.H. Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and
Steady State Enzyme Systems, second ed., Wiley-Interscience, New York, 1975.
[38] R.A. Peralta, A.J. Bortoluzzi, B. de Souza, R. Jovito, F.R. Xavier, R.A.A. Couto, A.
Casellato, F. Nome, A. Dick, L.R. Gahan, G. Schenk, G.R. Hanson, F.C.S. de Paula,
E.C. Pereira-Maia, S.D. Machado, P.C. Severino, C. Pich, T. Bortolotto, H. Terenzi,
E.E. Castellano, A. Neves, M.J. Riley, Inorg. Chem. 49 (2010) 11421.
[39] Y. Simon-Manso, J. Phys. Chem. A. 109 (2005) 2006.
[40] C. Hansch, A. Leo, R.W. Taft, Chem. Rev. 91 (1991) 165.
[41] J. Clayden, N. Greeves, S. Warren, Organic Chemistry, second ed., Oxford
University Press, Oxford, 2012.
[42] J. Xia, Y.B. Shi, Y. Zhang, Q. Miao, W.X. Tang, Inorg. Chem. 42 (2003) 70.
[43] M. Livieri, F. Mancin, U. Tonellato, J. Chin, Chem. Commun. (2004) 2862.
[44] E. Kimura, Y. Kodama, T. Koike, M. Shiro, J. Am. Chem. Soc. 117 (1995) 8304.
[45] E. Kimura, I. Nakamura, T. Koike, M. Shionoya, Y. Kodama, T. Ikeda, M. Shiro, J.
Am. Chem. Soc. 116 (1994) 4764.
4. Conclusion
The
complexes
[Zn2(H2L4)(H2O)2](ClO4)
and
[Zn2(H2L5)(CH3CO2)(H2O)](PF6)2possess properties analogous to
hydrolase metalloenzymes [19]. The latter complex has kinetic
properties and a kinetically relevant pKa (6.6) similar to those re-
ported for a number of related complexes and it is proposed that
the active nucleophile is a Zn(II)–OH moiety [36]. Structurally
[Zn2(H2L4)(H2O)2]+ presents a crowded active site motif and a
more basic kinetically relevant pKa (9.4). For this complex mono-
dentate coordination of the substrate followed by attack of the
nucleophile coordinated to the second metal ion is proposed.
Acknowledgement
[46] T. Koike, S. Kajitani, I. Nakamura, E. Kimura, M. Shiro, J. Am. Chem. Soc. 117
(1995) 1210.
[47] C. Bazzicalupi, A. Bencini, E. Berni, A. Bianchi, V. Fedi, V. Fusi, C. Giorgi, P.
Paoletti, B. Valtancoli, Inorg. Chem. 38 (1999) 4115.
This work was funded by a grant from the Australian Research
Council (DP0986613).
[48] S.A. Li, D.X. Yang, D.F. Li, J. Huang, W.X. Tang, New J. Chem. 26 (2002) 1831.
[49] D. Yang, S. Li, D. Li, J. Xia, K. Yu, W. Tang, Dalton Trans. (2002) 4042.
[50] L.V. Penkova, A. Maciag, E.V. Rybak-Akimova, M. Haukka, V.A. Pavlenko, T.S.
Iskenderov, H. Kozlowski, F. Meyer, I.O. Fritsky, Inorg. Chem. 48 (2009) 6960.
[51] L.M. Berreau, Adv. Phys. Org. Chem. 41 (2006) 79.
References
[1] M.A. Sogorb, E. Vilanova, Toxicol. Letts. 128 (2002) 215.
[2] C.J. Jackson, P.D. Carr, J.W. Liu, S.J. Watt, J.L. Beck, D.L. Ollis, J. Mol. Biol. 367
(2007) 1047.
[3] F.M. Raushel, Curr. Opinion Microbiol. 5 (2002) 288.
[4] F. Ely, J.L. Foo, C.J. Jackson, L.R. Gahan, D. Ollis, G. Schenk, Curr. Top. Biochem.
Res. 9 (2007) 63.
[5] D.W. Christianson, J.D. Cox, Annu. Rev. Biochem. 68 (1999) 33.
[6] D.P. Dumas, S.R. Caldwell, J.R. Wild, F.M. Raushel, J. Biol. Chem. 264 (1989)
19659.
[7] C. Jackson, P.D. Carr, H.K. Kim, J.-W. Liu, P. Herrald, N. Mitic, G. Schenk, C.A.
Smith, D.L. Ollis, Biochem. J. 397 (2006) 501.
[8] C.J. Jackson, J.L. Foo, H.K. Kim, P.D. Carr, J.W. Liu, G. Salem, D.L. Ollis, J. Mol. Biol.
375 (2008) 1189.
[9] H. Yang, P.D. Carr, S.Y. McLoughlin, J.-W. Liu, I. Horne, X. Qiu, C.M.J. Jeffires, R.J.
Russell, J.G. Oakeshott, D.L. Ollis, Protein Eng. 16 (2003) 135.
[10] M.M. Benning, H. Shim, F.M. Raushel, H.M. Holden, Biochemistry 40 (2001)
2712.
[11] M.M. Benning, J. Kuo, F. Raushel, H.M. Holden, Biochemistry 34 (1995) 7973.
[12] C.J. Jackson, H.K. Kim, P.D. Carr, J.-W. Liu, D.L. Ollis, Biochim. Biophys. Acta
1752 (2005) 56.
[13] L.R. Gahan, S.J. Smith, A. Neves, G. Schenk, Eur. J. Inorg. Chem. 19 (2009) 2745.
[14] A.J. Kirby, Angew. Chem. Int. Ed. 35 (1996) 707.
[15] M. Komiyama, J. Biochem. 118 (1995) 665.
[16] J.K. Bashkin, Curr. Opinion Chem. Biol. 3 (1999) 752.
[17] G.Q. Feng, J.C. Mareque-Rivas, N.H. Williams, Chem. Commun. (2006) 1845.
[18] G.Q. Feng, D. Natale, R. Prabaharan, J.C. Mareque-Rivas, N.H. Williams, Angew.
Chem. Int. Ed. 45 (2006) 7056.
[19] N. Mitic, S.J. Smith, A. Neves, L.W. Guddat, L.R. Gahan, G. Schenk, Chem. Rev.
106 (2006) 3338.
[20] K. Hadler, N. Mitic, F. Ely, G. Hanson, L. Gahan, J. Larrabee, D. Ollis, G. Schenk, J.
Am. Chem. Soc. 131 (2009) 11900.
[21] G. Schenk, T.W. Elliott, E.W.W. Leung, N. Mitic, L.E. Carrington, L.R. Gahan, L.W.
Guddat, BMC Struct. Biol. 8 (2008), http://dx.doi.org/10.1186/1472.
[22] M. Monroe, Molecular Weight Calculator, Version 6.45, 2004, http://
www.alchemistmatt.com/.
[23] R.T. Paine, Y.C. Tan, X.M. Gan, Inorg. Chem. 40 (2001) 7009.
[24] S. Striegler, M. Dittel, Inorg. Chem. 44 (2005) 2728.
[52] C. He, S.J. Lippard, J. Am. Chem. Soc. 122 (2000) 184.
[53] M. Yashiro, H. Kaneiwa, K. Onaka, M. Komiyama, Dalton Trans. (2004) 605.
[54] N.V. Kaminskaia, C. He, S.J. Lippard, Inorg. Chem. 39 (2000) 3365.
[55] M. Arca, A. Bencini, E. Berni, C. Caltagirone, F.A. Devillanova, F. Isaia, A. Garau,
C. Giorgi, V. Lippolis, A. Perra, L. Tei, B. Valtancoli, Inorg. Chem. 42 (2003) 6929.
[56] M. Jarenmark, E. Csapo, J. Singh, S. Wockel, E. Farkas, F. Meyer, M. Haukka, E.
Nordlander, Dalton Trans. 39 (2010) 8183.
[57] H. Sakiyama, Y. Igarashi, Y. Nakayama, M.J. Hossain, K. Unoura, Y. Nishida,
Inorg. Chim. Acta 351 (2003) 256.
[58] Y.-H. Chiu, J.W. Canary, Inorg. Chem. 42 (2003) 5107.
[59] R.C. diTargiani, S. Chang, M.H. Salter, R.D. Hancock, D.P. Goldberg, Inorg. Chem.
42 (2003) 5825.
[60] J.C. Mareque-Rivas, R. Prabaharan, S. Parsons, Dalton Trans. (2004) 1648.
[61] Y.-H. Chiu, G.J. Gabriel, J.W. Canary, Inorg. Chem. 44 (2005) 40.
[62] Y. Yoshikawa, K. Kawabe, M. Tadokoro, Y. Suzuki, N. Yanagihara, A. Nakayama,
H. Sakurai, Y. Kojima, Bull. Chem. Soc. Jpn. 75 (2002) 2423.
[63] M. Yashiro, R. Kawahara, J. Biol. Inorg. Chem. 9 (2004) 914.
[64] I. Bertini, C. Luchinat, M. Rosi, A. Sgamellotti, F. Tarantelli, Inorg. Chem. 29
(1990) 1460.
[65] A. Neves, M. Lanznaster, A.J. Bortoluzzi, R.A. Peralta, A. Casellato, E.E.
Castellano, P. Herrald, M.J. Riley, G. Schenk, J. Am. Chem. Soc. 129 (2007) 7486.
[66] M. Lanznaster, A. Neves, A.J. Bortoluzzi, V.V.E. Aires, B. Szpoganicz, H. Terenzi,
P.C. Severino, J.M. Fuller, S.C. Drew, L.R. Gahan, G.R. Hanson, M.J. Riley, G.
Schenk, J. Biol. Inorg. Chem. 10 (2005) 319.
[67] P. Karsten, A. Neves, A.J. Bortoluzzi, M. Lanznaster, V. Drago, Inorg. Chem. 41
(2002) 4624.
[68] G. Schenk, R.A. Peralta, S.C. Batista, A.J. Bortoluzzi, B. Szpoganicz, A.K. Dick, P.
Herrald, G.R. Hanson, R.K. Szilagyi, M.J. Riley, L.R. Gahan, A. Neves, J. Biol. Inorg.
Chem. 13 (2008) 139.
´
[69] G. Parkin, Chem. Rev. 104 (2004) 699.
[70] E. Kimura, Curr. Opinion Chem. Biol. 4 (2000) 207.
[71] B. Bauer-Siebenlist, F. Meyer, E. Farkas, D. Vidovic, J.A. Cuesta-Seijo, R. Herbst-
Irmer, H. Pritzkow, Inorg. Chem. 43 (2004) 4189.