Synthesis and reactivity of a C3-symmetric trinuclear zinc(ii) hydroxide catalyst efficient at phosphate diester transesterification

Article abstract of DOI:10.1039/b706386e

Inspired by trinuclear Zn(ii) sites in enzymatic systems, a ligand system containing three preorganized (2-pyridyl)methyl piperazine moieties anchored onto a rigid C₃-symmetric triphenoxymethane platform has been developed for preorganizing thr

Full text of DOI:10.1039/b706386e

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Synthesis and reactivity of a C3-symmetric trinuclear zinc(II) hydroxide
catalyst efficient at phosphate diester transesterification†‡
Ranjan Mitra, Matthew W. Peters and Michael J. Scott*
Received 27th April 2007, Accepted 14th June 2007
First published as an Advance Article on the web 18th July 2007
DOI: 10.1039/b706386e
Inspired by trinuclear Zn(II) sites in enzymatic systems, a ligand system containing three preorganized
(2-pyridyl)methyl piperazine moieties anchored onto a rigid C3-symmetric triphenoxymethane platform
has been developed for preorganizing three zinc ions into an environment conducive to intramolecular
interaction. Zinc(II) binding by this ligand has been analyzed by means of potentiometric
measurements in 50% (v/v) CH3CN–H2O solutions. Subsequently a C3-symmetric trinuclear Zn(II)
hydroxide complex of the C3-symmetric ligand was synthesized and fully characterized using NMR
spectroscopy and X-ray crystallography. This complex induces a 16 900-fold rate enhancement in the
catalytic cyclization of the RNA model substrate, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP,
pH 6.7, 25 ◦C) over the uncatalyzed reaction with multiple catalyst turnovers. The observed differences
in the pH-rate profile can be attributed to the varying concentration of various trinuclear zinc species.
The trinuclear Zn(II) catalyst exhibits a higher hydrolytic activity compared to its mononuclear
analogue. The reactivity and structural features of this trinuclear Zn(II) complex will be discussed.
The phosphodiesters that form the structural backbone of nucleic
acids are extremely resistant to hydrolytic cleavage. The estimated
half-life of RNA is 110 years and that of DNA is in the range of
10–100 billion years.1,2 As a consequence, nature has to use various
enzymes to accelerate the hydrolysis and to enable the processing
of nucleic acids under physiological conditions. Most phosphodi-
esterases contain two or even three divalent metal ions close to
each other, which work synergistically as a single unit.3–6 The well
studied dinuclear zinc phosphatase includes phosphotriesterase7,8
and alkaline phosphatase.9,10 Enzymes that incorporate three zinc
centers are also known such as phospholipase C11–13 and nuclease
P1.14,15 In these systems, there are two closely associated Zn(II)
centers together with a more distant Zn(II) ion. For example,
while the separation of the principal dizinc unit in phospholipase
enzymes.2,18–25 By using a variety of spacers, two or more simple
ligands, each capable of binding one zinc ion, can be linked
together by groups ranging from simple aliphatic chains to more
complex groups such as calix[n]arenes.26–41 Other approaches have
focused on the large macrocycles that encompass two or more
metal ions into a single framework,42–52 oftentimes producing very
effective structural models for these metallohydrolases.53
Synthetic analogues that mimic all aspects of these zinc enzymes
i.e. structure, function, and mechanism, are yet to be obtained.
Many of the structural models are inactive for catalysis,20 while in
functional models it is frequently difficult to discern the identity
of the competent catalysts since the molecules are often prepared
in situ.20 Thus, to have a better understanding of the catalytic
mechanism, it could be advantageous to combine both structural
and functional information into a single system. With these issues
in mind, a simple ligand capable of preorganizing three metal ions
into an environment conducive to intramolecular interaction was
designed using the C3-symmetric triphenoxymethane as a plat-
form.54 In this paper we describe the synthesis of ligand 5 using the
C3-symmetric triphenoxymethane platform, the formation of the
corresponding Zn(II) complex and its catalytic activity in the cleav-
age of the phosphate diester bond of an RNA model substrate.
˚
C is 3.3 A, the distances between these centers and the third zinc
3
˚
center are 4.7 and 6.0 A. Likewise the corresponding separations
in P1 nuclease are 3.2, 4.7 and 5.8 A. It is postulated that in
3,14
˚
P1 nuclease, the hydroxide bridging the two closely associated
Zn(II) centers attacks the phosphate substrate that is bound to the
third, more distant, Zn(II) ion in a bidentate mode.14,15 Despite
extensive investigations, however, details of the mechanism of this
and other metallohydrolases remain controversial. One crucial
aspect under debate is the identity and the exact binding mode
of the nucleophile.16,17 Also, the function of a third metal ion in
close proximity of a dinuclear metal cluster in these enzymes is
not yet fully understood. A diverse assortment of ligands have
been employed to model various aspects of multinuclear zinc
Results and discussion
Ligand synthesis and structural characterization of 5 and 7
To develop a synthetic model for the trinuclear zinc enzymes, three
nitrogen based ligands, each capable of binding one zinc ion, can
be attached to a platform capable of reorganizing these moieties
into an environment conducive to intramolecular interactions
between the three bound metal ions. Previous work with a triphe-
noxymethane platform has shown that, when all the three phenol
oxygens are substituted, the conformation with these oxygen atoms
“all up” relative to the central methine hydrogen of the platform
Department of Chemistry and Center for Catalysis, University of Florida,
Gainesville, FL, 32611-7200, USA. E-mail: mjscott@chem.ufl.edu; Fax: +1
352 392-3255; Tel: +1 352 846-1165
† CCDC reference numbers 645249 and 645250. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/b706386e
‡ Electronic supplementary information (ESI) available: Synthetic proce-
dures and characterization data for 8, 9, 10, 11, 1H NMR spectra of 7 in
the presence of buffer at pH = 6.7. See DOI: 10.1039/b706386e
3924 | Dalton Trans., 2007, 3924–3935
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exclusively exists both in the solid state and in solution.54 Extended
arms can easily be secured to the phenolic oxygens (Scheme 1).
NMR spectral data and the X-ray crystal structure of 5 (Fig. 1)
illustrate that indeed, the conformation of the platform is “all up”
and that the three metal binding sites are preorganized and poised
for modeling biological trinuclear active sites.
Scheme 1 Synthesis of ligand 5. Reagents and conditions: (i) 3.3 equiv.
BrCH2COOEt, 4 equiv. Cs2CO3, acetone, reflux (ii) 6 equiv. LiAlH4, Et2O
(iii) 4 equiv. p-CH3C6H4SO2Cl, pyridine (iv) 30 equiv. 1-[(2-pyridyl)methyl]
piperazine, 10 equiv. Na2CO3, acetonitrile, 5 d reflux.
Fig. 2 Depiction of the solid-state structure of 7 (a) Side view with hy-
drogen atoms and perchlorate anions omitted for clarity (30% probability
ellipsoids, carbon atoms arbitrary radii). Primed and unprimed atoms are
related by a 3-fold symmetry axis. (b) Cartoon of the top view of the core.
Table 1 Selected interatomic distances and angles for 7·3(Me)2CO
Bond angles/◦
˚
Bond lengths/A
Zn1–O1
Zn1–O1ꢀꢀ
Zn1–N1
Zn1–N2
Zn · · · Zn
1.925(4)
1.903(4)
2.055(5)
2.143(5)
3.435
N1–Zn1–N2
N1–Zn1–O1
N1–Zn1–O1ꢀꢀ
N2–Zn1–O1ꢀꢀ
O1–Zn1–O1ꢀꢀ
N2–Zn1–O1
81.0(2)
111.7(2)
127.28(19)
104.3(2)
109.4(2)
121.01(19)
[N2–Zn1–O1]. Three perchlorate anions balance the charge on
the complex. Similar coordination geometries have been observed
in a few N-methylpiperazine and morpholine based ligands.58 Al-
though, the Zn3(OH)3 structural core has been obtained previously
from self-assembly reactions with small ligands, to the best of our
knowledge, hydrolytic cleavage of phosphate diester bonds using
these complexes has not yet been reported.55–57
Fig. 1 The X-ray crystal structure of 5 drawn with 30% ellipsoids (carbons
The NMR spectrum of the ligand undergoes significant changes
upon incorporation of zinc ions into the ligand (see ESI‡, Fig. S1,
S2). The resonance for the central methine proton shifts downfield
from 6.52 ppm in 5 to 7.10 ppm in 7. The coordination of zinc
by the ligand arms forms new cyclic structures that result in the
splitting and shifting of various peaks, especially those associated
with the (2-pyridyl)methyl piperazine moiety. Fluctuations in the
linker moieties connecting these groups to the triphenoxymethane
platform and within the coordination sphere of the metal cause
with arbitrary radii). Hydrogen atoms omitted for clarity.
Addition of three equivalents of Zn(ClO4)2·6H2O to a solution
of 5, afforded the trinuclear zinc(II) complex, 7. The solid-state
structure of 7, as determined by X-ray crystallography, is shown
in Fig. 2 and selected bond lengths and angles are given in Table 1.
In the solid state, the molecule possesses rigorous C3-symmetry
˚
with a distance of 3.43 A between the zinc centers. The three
zinc cations are assembled into a distorted six-membered ring
connected together by three bridging hydroxyl groups, and the
angle about the zinc and hydroxides differ significantly [O1–Zn1–
O1ꢀ 109.4(2)◦ and Zn1–O1–Zn1ꢀ 127.6(2)◦]. The metal hydroxide
1
these split peaks to appear broadened in the spectra. H NMR
spectroscopy suggests that on the NMR time scale a C3-symmetric
core is maintained in solution, under the conditions (pH 6.7, 50%
(v/v) CD3CN–D2O, buffer, [7] = 7.5 mM) used for the kinetic
experiments described below. The species distribution diagram
depicted in Fig. 6, (vide infra), however, indicated the presence of
various trinuclear zinc species in solution at and around pH 6.7,
suggesting a proton exchange reaction occurs quite fast in these
solutions.59 All solutions remained clear during the time of the
distances are typical for a Zn3(OH)3 core and differ slightly
ꢀꢀ
[1.925(4) A, Zn1–O1; 1.903(4) A, Zn1–O1 ].55–57 The zinc nitrogen
˚
˚
˚
separations are asymmetric with a bond length of 2.055(5) A to
˚
the pyridyl nitrogen and 2.143(5) A to the piperazine nitrogen.
The metal maintains a highly distorted tetrahedral geometry with
bond angles ranging from 81.0(2)◦ [N1–Zn1–N2] to 127.28(19)◦
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kinetic experiments and no peak corresponding to the free ligand
was observed. After prolonged periods at pH 6.7, however, peaks
corresponding to the free ligand do appear in the 1H NMR
spectrum indicating that the metals can be slowly leached out
of the ligand. After about 24 h in the absence of HPNP, the system
reached chemical equilibrium with the buffer solution containing
complex 7 and free ligand 5 in a ∼ 6 : 1 ratio, but under the catalytic
condition, within the first 5% conversion of the reactant, the core
of 7 is stable with very little free Zn(II) or free ligand in solution
(see ESI‡, Fig. S3–S6). Thus the method of initial rates (< 5%
conversion) was used to study these reactions (vide infra).
Fig. 3 shows the variation of the ratio of free ligand (5) to
trinuclear zinc complex (7) as a function of pH after 30 min of
mixing. The change in the ligand concentration was followed by
measuring the peak area at 8.44 ppm corresponding to a pyridyl
proton of the free ligand. The peak area was normalized relative
to the peak area of the same proton, at 8.62 ppm, in the trinuclear
zinc complex 7. A gradual decrease in the ratio of free ligand (5) to
trinuclear zinc complex (7) was observed as the pH was raised from
pH 5.87 to pH 6.7. Further increasing the pH to 7.01 produced a
slight increase in the ratio. Under these conditions, the depletion
of Zn may be due to the formation of Zn(II)-MES (MES = 2-
morpholinoethanesulfonic acid) buffer complex.60 In the absence
of a buffer the 50% (v/v) CD3CN–D2O solution of 7 is stable for
weeks (see ESI‡, Fig. S4). At higher pH values, the Zn(II) complex
is increasingly unstable owing to the formation of insoluble zinc
hydroxide.
Fig. 4 Temperature-dependent 1H NMR spectra of the aromatic region
of 7. The reaction mixture contains 7 (7.5 mM), MES buffer (22.5 mM) in
1 : 1 (CD3CN–D2O) at pH = 6.7.
Solution equilibria
While the X-ray crystallographic results provide structural insights
for the various complexes, knowledge of the species distribution
in solutions is crucial for understanding any trends in hydrolytic
reactivity of artificial metallohydrolases. To investigate the re-
lationship between the pH dependence of these reactions and
the structures of the aquo-trizinc complexes in solution, the pKa
values of the ligand, 5, (Table 2), as well as the stability constants
of its zinc complexes and the pKa values of zinc bound water
molecules in these complexes (Table 3), were determined by pH
titrations. Buffer was included in the solutions for all of the NMR
experiments but it was omitted from the potentiometric titrations
due to the nature of the experiment.
Table 2 Protonation constants of the ligand 5 at 25 ◦C; I = 0.1 M KCl
Species
Log b
pKa
[LH6]6+
[LH5]5+
[LH4]4+
[LH3]3+
[LH2]2+
[LH]+
37.96
34.15
30.20
24.10
16.83
9.23
3.81
3.95
6.10
7.27
7.60
9.23
Fig. 3 Variation of the ratio of free ligand (5) to trinuclear zinc complex
(7) as a function of pH.
Table 3 Stability constants of the Zn(II) complexes with 5 at 25 ◦C; I =
Fig. 4 shows the temperature dependence of the 1H NMR
spectrum of the aromatic region of 7 in 50% (v/v) CD3CN–D2O at
pH 6.7 (peaks corresponding to the aliphatic region are not shown
as they are masked by the presence of a large excess of MES buffer).
Increasing the temperature from 10 C to 55 C induced only a
minor sharpening of the peaks suggesting that on the NMR time
scale the proton exchange between various species in solution is
quite fast in this temperature range. The compound precipitates
out of solution below 10 ◦C thus restricting the experiment within
a span of 45 ◦C.
0.1 M KCl
Species
Log b
pKa
[Zn3LH3]9+
[Zn3LH2]8+
[Zn3LH]7+
36.09
31.46
25.93
19.27
12.77
5.88
4.63
5.53
6.66
6.50
6.89
7.63
◦
◦
[Zn3L]6+
[Zn3L(OH)]5+
[Zn3L(OH)2]4+
[Zn3L(OH)3]3+
−1.75
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Ligand protonation constants.
nitrogen of the piperazinium ion. The titration data were analyzed
for the following equilibria. The protonation constants K1–K6 are
defined as follows:
The protonation constant of the ligand was determined by
potentiometric titration of 6 (1 mM) with 0.1 M KOH, I =
0.1 M (KCl) at 25 ◦C. Studies were carried out in 50% (v/v)
CH3CN–H2O mixtures due to the low water solubility of the
ligand and the metal complex in pure water. The pH titration
curve is shown in Fig. 5. Although the ligand has as many as
nine protonation sites (three per side arm), only six measurable
deprotonation processes were found in the pH range 2.5 to 11.5.
The nitrogen atoms on the pyridyl groups are less basic than those
of the aliphatic amines because the former has more s character
than the latter.61 It has also been observed that as the separation
between the protonation sites becomes smaller or as the charge on
the ligand increases, the successive protonation of the less basic
nitrogens becomes progressively more difficult till it reaches a level
where protonation is barely accessible in dilute aqueous solutions
of low ionic strength.62
H+ + L ꢀ HL+ K1 = [HL+]/([H+][L])
H+ + HL+ ꢀ H2L2+ K2 = [H2L2+]/([H+][HL+])
H+ + H2L2+ ꢀ H3L3+ K3 = [H3L3+]/([H+][H2L2+])
H+ + H3L3+ ꢀ H4L4+ K4 = [H4L4+]/([H+][H3L3+])
H+ + H4L4+ ꢀ H5L5+ K5 = [H5L5+]/([H+][H4L4+])
H+ + H5L5+ ꢀ H6L6+ K6 = [H6L6+]/([H+][H5L5+])
The values of the protonation constants obtained are in agree-
ment with the values of various similar amines in the literature
(Table 4).64 While comparing the values, however, it should be
kept in mind that hydrogen bonding with pyridine nitrogen and
various other interactions, such as substituent effects where by
tertiary amines gain electron density by aliphatic substituents and
suffer from a withdrawal of electron density by the picolyl group,
have a profound effect on the protonation constant of the ligand.
Metal complexation in aqueous solution
Titration of the protonated ligand in the presence of 1, 2 and 3
equivalents of Zn(II) were analyzed in batch calculations in which
all titrations curves were simultaneously fitted with one model
(Fig. 6). The accessible pH range has been limited in some of
these experiments due to the formation of precipitates. Titrations
were stopped as soon as a steady drift was noted in the mV meter
reading, indicative of the initiation of the precipitation process.
Formation of an insoluble species was observed for pH values
higher than pH 8.2, 8.0 and 7.8 for 1 : 1, 1 : 2, and 1 : 3 (L–M)
ratios, respectively.
Table 4 pKa values of similar amines in aqueous solution at 25 ◦C; I =
0.1 M KCl
Log Ka
Amine
HL/H*L
8.61
H2L/HL*H
2.00
H3L/H2L*H
—
Fig. 5 Potentiometric titration curve of 6 with 0 (+), 1 (ꢀ), 2 (᭺) or 3 (D)
equiv. Zn(II) in 0.1 M KCl in 1 : 1 CH3CN–H2O at 25 ◦C.
If only three protons attached to the pyridyl groups are released
upon dissolving in a 50% (v/v) CH3CN–H2O solution of low
ionic strength (0.1 M), the initial hydrogen ion concentration will
be three times that of the ligand concentration and hence the
theoretical initial pH of the solution can be computed as follows:63
pH = −log [(3 × mmol of ligand)/total volume (mL)] = −log [(3 ×
0.046)/50] = 2.56. This value is, in fact, very close to the observed
value of 2.53. The first three pKa values are thus below 2.5, and
were not considered in the equilibrium model for data analysis.
In the following three steps, one proton is removed from each
arm, and corresponds to the proton attached to the nitrogen of
the piperazinium ion closer to the pyridyl group. The final three
deprotonations again correspond to the dissociation of one proton
per sidearm, but the protons involved are attached to the other
8.91
9.46
—
—
5.91
2.73
9.71
8.13
5.59
4.18
—
—
a Values taken from reference 64
.
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of the stability constants of the trinuclear species [Zn3LH3]9+,
[Zn3LH2]8+, [Zn3LH]7+, [Zn3L]6+, [Zn3L(OH)]5+, [Zn3L(OH)2]4+
and [Zn3L(OH)3]3+. As often found in polyamine ligands with a
large number of amine donors,64 the mononuclear and dinuclear
complexes display a marked tendency to add a third metal to give
stable trinuclear Zn(II) species, which are largely prevalent in 50%
(v/v) CH3CN–H2O solutions containing metal and ligand in a
3 : 1 molar ratio, Fig. 6. Though the species distribution diagram
indicates the presence of various trinuclear zinc species in solution,
1
the H NMR spectra of 7 in 50% (v/v) CD3CN–D2O indicate a
highly symmetric structure. This suggests that the proton exchange
reaction between the various species is sufficiently fast in 50% (v/v)
CD3CN–D2O on the NMR time scale.59
The formation of [Zn3L]6+ species occurs at acidic pH and is
followed by the formation of mono-, di- and trihydroxo complexes
by successive deprotonation of the coordinated water molecules at
slightly acidic to alkaline pH values. The structure of trihydroxo
species, [Zn3L(OH)3]3+ should correspond to the structure of the
cation of 7 which was characterized by X-ray crystallography. In
this complex, the metal displays a coordination environment not
saturated by the ligand donors. All three zinc binding sites are
occupied by tetrahedral zinc(II) ions coordinated by the pyridyl
nitrogen and the piperazine nitrogen atoms from each arm of the
ligand. The remaining two coordination sites of the zinc ion are
filled by bridging hydroxides. The trinuclear Zn(II) species, [Zn3L]6+
exhibited low pKa values for the formation of mono-, di- and
Fig. 6 Species distribution diagram for 6 (0.92 mM) in the presence of
Zn(II) ions (2.76 mM) as a function of pH in 1 : 1 CH3CN–H2O at 25 ◦
C
with I = 0.10.
Accordingly, the calculations were carried out using data
obtained below their respective pH values. Best fit of the titration
data could be attained using the following equilibrium model
L + 3Zn2+ ꢀ Zn3L6+
KZn3L = [Zn3L6+]/[L][Zn2+]3
H+ + Zn3L6+ ꢀ Zn3LH7+
trihydroxo trinuclear complexes (pKa1 = 6.53, pKa2 = 6.88, pKa3
=
7.64). The first two pKa values are quite close to one another and
differ by only 0.35 units. The pKa value for deprotonation of the
first water molecule in the [Zn3L]6+ trinuclear complex is very low.
This behavior indicates a strong binding of the hydroxide ion in
[Zn3L(OH)]5+ and is generally ascribed to a bridging coordination
mode of OH between two metal centres.40,47,52 This hypothesis is
corroborated by the crystal structure of the [Zn3L(OH)3]3+ cation,
Fig. 2, which shows three zinc ions connected to each other by three
bridging hydroxides. The metal coordination spheres of the three
metal centers are not fulfilled by the ligand donors. Therefore,
they would provide efficient substrate binding and activation.
At the same time, facile deprotonation of metal-bound water
molecules occur from slightly acidic to neutral pH, giving rise to
Zn–OH groups as potential nucleophiles in hydrolytic reactions.
These features make the trinuclear Zn(II) complex 7 a promising
hydrolytic agent.
KZn3LH = [Zn3LH7+]/[H+][Zn3L6+]
8+
H+ + Zn3LH7+ ꢀ Zn3LH2
KZn3LH2 = [Zn3LH28+]/[H+][Zn3LH7+]
H+ + Zn3LH2 ꢀ Zn3LH3
8+
9+
KZn3LH3 = [Zn3LH39+]/[H+][Zn3LH2
Zn3L + H2O ꢀ Zn3L(OH)5+ + H+
]
8+
KZn3LOH = [Zn3L(OH)5+][H+]/[Zn3L]
Zn3L(OH)5+ + H2O ꢀ Zn3L(OH)2 + H+
4+
It is not possible to determine reaction mechanism based solely
on crystal structure data. Nevertheless, structural information,
when combined with kinetic data, can be very powerful for solving
reaction mechanisms.
KZn3L(OH)2 = ([Zn3L(OH)24+][H+])/[Zn3L(OH)5+]
Zn3L(OH)2 + H2Oꢀ Zn3L(OH)3 + H+
4+
3+
Transesterification of hydroxypropyl-p-nitrophenyl phosphate
(HPNP)
KZn3L(OH)3 = [Zn3L(OH)33+][H+]/[Zn3L(OH)2
]
4+
The occurrence of mononuclear and dinuclear species was
also considered. Using such an extended equilibrium model for
the curve-fitting procedure did not affect the overall standard
deviation, which suggests that the formation of these species
was negligible. This observation is consistent with the NMR
spectral data of 7 in 50% (v/v) CH3CN–H2O wherein formation
of mononuclear or dinuclear species was not observed even after
two weeks. The results of the fitting allowed the calculation
Complex 7 is insoluble in pure water and hence CH3CN was
added as a cosolvent.65 The catalytic activity of the trinuclear zinc
complex 7, towards the transesterification of HPNP was studied in
50% CH3CN–80 mM aqueous buffer at 25 ◦C. The pH dependence
for the transesterification of HPNP by 7 was studied in the pH
range of 6.4 to 7.2 and the optimum pH for the reaction was found
to be 6.7 (Fig. 7). The catalyst precipitated out of the reaction
mixture above pH 7.4. At pH 6.7, 5 mM of 7 induces a 16 900
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The binding affinity of 7 towards HPNP was studied by
measuring the rate of transesterification as a function of HPNP
concentration, to investigate the catalytic process in more detail,
at a fixed catalyst concentration. No saturation kinetics could
be observed for 7 with up to 7 equivalents of HPNP (pH 6.6)
indicating a low affinity for the substrate (Fig. 9). It is not possible
to determine kobs when [Zn(ClO4)2] is ≥ 0.8 mM at pH 6.7 in the
absence of ligand due to precipitation of Zn(II) hydroxide. With
0.4 mM Zn(ClO4)2, the value of kobs was found to be (1.2 0.1) ×
10−6 s−1, indicating that the catalytic contribution of free Zn(II)
towards the cleavage of HPNP in the presence of 7 is negligible.
Fig. 7 pH versus rate profile for transesterification of HPNP (2 mM)
catalyzed by 7 (5 mM) in acetonitrile–80 mM buffer 1 : 1 (v/v) at 25 ◦C.
fold rate enhancement in the catalytic cyclization of the RNA
model substrate, HPNP, compared to the uncatalyzed reaction,
corresponding to a reduction of the half-life from approximately
308 d (kuncat = (2.6
0.1) × 10−8 s−1, error limits are given
at the 95% confidence level unless otherwise stated)26,66 for the
uncatalyzed reaction to 26 min (kobs = (4.4 0.1) × 10−4 s−1). The
pH rate profile was then compared with the species distribution
diagram (Fig. 6) in order to identify the reactive species. Since
considerable concentration of [Zn3LH]7+, [Zn3L]6+, [Zn3L(OH)]5+
and [Zn3L(OH)2]4+ are present in the solution, in the pH range 6.4
to 7.2, any or all of these might be the active species catalyzing
the transesterification of HPNP. Thus, the optimum activity of the
catalyst at pH 6.7 may not be due to the change in concentration
of one particular species but is a consequence of the varying
concentration of the different trinuclear zinc species.
Fig. 9 Initial rate as a function of the substrate concentration for the
transesterification of HPNP.
To determine whether 7 acts as a catalyst for the transester-
ification of HPNP, reactions in the presence of 82 equivalents
of the substrate were followed by 31P NMR spectroscopy. A
progressive increase in the intensity of the signal corresponding
to the cyclic phosphate ester with a simultaneous decrease of
the HPNP signal was noted as outlined in Fig. 10. No other
signal, including that of inorganic phosphate, was observed even
after prolonged reaction times. In a separate experiment, catalytic
turnover was demonstrated by preparing a reaction mixture
consisting of 0.1 mM 7, 2 mM HPNP and 80 mM MES buffer
(pH 6.7). The absorbance at 400 nm produced by the release of
p-nitrophenolate ion was monitored over a two day period and
more than 10 turnovers of catalyst was noted. During the course of
the multiple turnover reaction, the catalyst activity does decrease
possibly due to product inhibition or catalyst decomposition as
previously discussed.
The effect of concentration of 7 on the reaction rate was then
investigated at pH 6.6. Within the concentration range explored
(1.0 to 5.0 mM) the observed pseudo first order rate constant (kobs),
for the transesterification of HPNP, exhibits a linear dependence
on the concentration of the complex. The second order rate
constant was determined as the slope of the linear plot of kobs
against catalyst concentration (k2 = (7.9 0.2) × 10−2 M−1 s−1,
Fig. 8).
Fig. 8 Dependence of the observed pseudo first order rate constant
on the concentration of trinuclear zinc(II) catalyst 7 at pH 6.6 in 50%
CH3CN–80 mM MES buffer (v/v) at 25 ◦C. [HPNP] = 2 mM.
Fig. 10 Stack plot of 31P NMR for the transesterification of HPNP
(38.56 mM) catalyzed by 7 (0.47 mM) at pH 6.7 after (b) 1 d (c) 3 d
(d) 5 d (e) 8 d. Spectrum (a) was taken before addition of the catalyst.
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Table 5 Rate constants for the transesterification of HPNP catalyzed by
Zn(II) complexes at 25 ◦
Potential catalytic mechanisms
C
To facilitate a better insight into the mechanism, phosphodi-
esterase activity of the trinuclear zinc complex 7 was compared
with that of a mononuclear zinc complex, prepared in situ by the
addition of one equivalent of Zn(ClO4)2·6H2O to a solution of
11 (Fig. 12). A progressive increase in the intensity of the signal
corresponding to the cyclic phosphate ester with a simultaneous
decrease of the HPNP signal can be noted in Fig. 13. No other
signal, including that of inorganic phosphate, was observed even
after prolonged reaction times, indicating that the cyclic phosphate
ester is not further hydrolyzed by the mononuclear zinc complex.
The trinuclear Zn(II) catalyst exhibits a higher hydrolytic activity
as compared to its mononuclear analogue as can be realized
qualitatively by comparing Fig. 10 and Fig. 13. The mononuclear
analog is, however, more active than free Zn(II) ions.
Catalysta
pH
k2× 102/M−1 s−1
Ref.
7
6.7
7.0
7.0
7.4
7.0
7.6
7.6
7.6
7.6
7.6
7.9
1.5
4300
1700
290
0.21
25
1.1
This work
Zn(12)
Zn2(13)
Zn2(13)
Zn3(14)
Zn(15)
Zn2(16)
Zn2(17)
Zn2(18)
Zn2(19)
23
23
23
23
29
29
29
29
29
0.89
0.58
a For structures of these complexes see Fig. 11.
With ligands adept at forming trimetallic complexes, the
orientation and flexibility of the metal binding groups impacts
substrate binding. Reinhoudt and coworkers investigated the
transesterification of HPNP catalyzed by the co-operative action
of multiple Zn(II) centers tethered to the upper rim of a semi-
flexible calix[4]arene.26,66 The trizinc calix[4]arene complex exhib-
ited saturation kinetics, and the second order rate constant, k2
for the transesterification of HPNP by this catalyst at pH 7 is
2.9 M−1 s−1. Strong substrate binding to the Zn(II) complex has the
effect of increasing the second order rate constant. In the case of
7, substrate binding appears to be impeded either by the crowded
nature of the trinuclear cluster or possibly by the presence of
bridging hydroxides between the metal centers, consequently, the
value for k2 is low. The bound hydroxides may lower the substrate
binding affinity of the catalyst by diminishing the Lewis acidity
of the Zn(II) centers.40,47,52 Nevertheless, once bound, HPNP is
rapidly converted to the product by 7. Table 5 summarizes the
second order rate constants (k2/M−1 s−1) for the transesterification
of HPNP catalyzed by several different Zn(II) complexes (Fig. 11).
Fig. 12 Structure of ligand 11.
Fig. 13 Stack plot of 31P NMR for the transesterification of HPNP
(38.56 mM) catalyzed by a mixture of 11 (1.41 mM) and Zn(ClO4)2·6H2O
(1.41 mM) at pH 6.7 after (b) 1 d (c) 3 d (d) 5 d (e) 8 d. Spectrum (a) was
taken before addition of 11 and Zn(ClO4)2·6H2O.
In the trinuclear zinc complex, the three Zn(II) ions are kept
in close proximity by use of a hydroxide bridging ligand.40,47,52
The bridging hydroxide group presumably shields the electrostatic
interactions between the Zn(II) ions and allows the cations to be
drawn relatively close together in a complex of greatly enhanced
activity. This high density of positive charge at 7 is ideal for
providing electrostatic stabilization of the transition state for
cleavage of HPNP relative to the reactant state because there is
a net increase in negative charge on proceeding from the reactant
to the transition state.
As discussed above, though any or all of the following species
([Zn3LH]7+, [Zn3L]6+, [Zn3L(OH)]5+ and [Zn3L(OH)2]4+) might be
the active species catalyzing the transesterification of HPNP, the
pH-rate profiles for metal-ion catalyzed cleavage of RNA and
RNA analogues frequently track the formation of metal ion
Fig. 11 Structure of ligands mentioned in Table 5.
Although a remarkable rate of acceleration in the transes-
terification of HPNP has been observed, no significant activity
for the cleavage of the internucleosidic phosphodiester bonds
of diribonucleoside monophosphate diester has been found for
complex 7 under a variety of conditions. These results indicate that
stabilization of the leaving group indeed plays an important part in
the hydrolytic cleavage of phosphate diester bonds in nucleotides
like RNA and DNA. Moreover, the trinuclear cluster might be
sterically too encumbered for the efficient substrate binding.
3930 | Dalton Trans., 2007, 3924–3935
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hydroxide complexes. A hydroxide (or water) spanning two zinc
ions has been detected crystallographically in many hydrolases
and is often considered as the active nucleophile, but it can be
expected to exhibit rather low nucleophilicity if coordinated in a
tightly bridging form.3,14,15,67 Thus, it has been suggested that upon
substrate binding a shift of the bridging hydroxide to a terminal
position occurs prior to attack on the coordinated substrate.68,69
As discussed above, in 7 three metal ions are held at a short
distance, and at the same time, the ligand donors poorly saturate
the coordination sphere of the metal ions. These structural features
would favor a bridging interaction mode of the phosphate ester.70
Substrate interaction with the electrophilic metal centers promotes
the nucleophilic attack of a Zn–OH function and thus enhances
the rate of hydrolytic process, as found in several synthetic
Zn(II) complexes.23,26,71 The l-1,3 bridged phosphoester unit has
frequently been postulated as the preferred substrate binding mode
in various metallophosphoesterases. A recent crystal structure
of native Eschericia coli alkaline phosphatase complexed with
The trinuclear Zn(II) complex exhibits a higher hydrolytic
activity as compared to its mononuclear analogue. Although 7
efficiently catalyzes the transesterification of HPNP, it is not cat-
alytically active in the hydrolysis of ethyl p-nitrophenyl phosphate
demonstrating that the b-hydroxyl group of HPNP is essential
for hydrolysis. Based on the experimental observations, the metal
catalyzed facile intramolecular transesterification of HPNP may
either involve the participation of the bridging hydroxide as
a general base or the nucleophilic attack by the deprotonated
hydroxy group of the substrate.
Experimental
General methods
All reagents and solvents were of analytical grade and were
used without purification, unless otherwise noted. Aqueous
solutions were prepared using 18 MX Millipore deionized
water. 4-tert-butyl-2-methylphenol, 2-morpholinoethanesulfonic
acid (MES), N-(2-hydroxyethyl)piperazine-Nꢀ-(2-ethanesulfonic
acid) (HEPES) and Zn(II) perchlorate hexahydrate were pur-
chased from Sigma–Aldrich. The barium salt of 2-hydroxypropyl-
4-nitrophenyl phosphate (HPNP)79 and 1-[(2-pyridyl)methyl]
inorganic phosphate shows the phosphate anions bridging the two
72–75
˚
Zn(II) ions, which lie 3.94 A apart from each other.
Similarly,
the X-ray crystal structure of the complex of PLCBC (phospho-
lipase C from Bacillus cereus) with a competitive inhibitor (a
phospholipid analog) revealed that the phosphate binds to all three
1
piperazine80 were prepared following literature methods. All H,
Zn(II) ions by replacing two of the zinc-bound water molecules in
13C and 31P NMR spectra were recorded on a Varian VXR-300 or
Mercury-300 spectrometer at 299.95, 75.4 and 121.42 MHz for the
proton, carbon and phosphorus channels, respectively. Elemental
analyses were performed by Complete Analysis Laboratories, Inc.
in Parsippany, NJ. IR spectra were recorded as KBr discs on a
Bruker Vector-22 instrument at a resolution of 2 cm−1. The pH me-
ter used for adjustment of buffered solution was calibrated daily.
13,76,77
the native PLCBC
.
Although 7 efficiently catalyzes the transesterification of HPNP,
it is not catalytically active in the hydrolysis of ethyl p-nitrophenyl
phosphate demonstrating that the b-hydroxyl group of HPNP
is essential for hydrolysis. Based on the above discussion, the
high activity in HPNP transesterification observed for our trin-
uclear Zn(II) complex may be explained by considering that the
phosphate ester would interact with the Zn(II) centers, resulting
in substrate activation. The subsequent facile intramolecular
transesterification of the phosphate diester bond may either
involve deprotonation of the hydroxy group by the bridging
hydroxide that acts as a general base or the direct coordination
of the substrate alcoholic function to the metal ion and the
subsequent nucleophilic attack by the deprotonated hydroxy group
of the substrate.78
Synthesis of the compounds
Detailed synthesis of 8, 9, 10 and 11 are reported in the ESI‡.
Tris(3-methyl-5-tert-butylphenoxy)methane (1)
Using an aldehyde condensation method for tris(3,5-tert-
butyl-2-hydroxyphenyl)methane,54,81 a 50 mL methanol solu-
tion of 10.66 g (64.9 mmol) of 2-methyl-4-tert-butylphenol,
and 5.33
g (27.7 mmol) 2-carboxaldehyde-6-methyl-4-tert-
Conclusions
butylphenol, chilled to 0◦ C in an ice bath was acidified to
saturation with ∼25 mL of thionyl chloride. The solution was
allowed to warm up to room temperature and then stirred
overnight, during which an off-white product, 1, precipitated from
the solution. The solid was filtered from the reaction mixture and
washed with methanol to afford 9.68 g (70%) of clean 1. IR: m
[cm−1] 3454 (O–H). 1H NMR ((CD3)2SO): d = 1.08 (s, 27 H, Ar–
C(CH3)3), 2.11 (s, 9 H, Ar–CH3), 6.60 (s, 1 H, CH), 6.70 (d,
4J(H,H) = 1.9 Hz, 3 H, Ar–H), 6.84 (d, 4J(H,H) = 1.9 Hz, 3 H,
Ar–H). 13C NMR ((CD3)2SO): d = 17.2 (Ar–C(CH3)3), 31.3 (Ar–
C(CH3)3), 33.5 (Ar–CH3), 36.0 (CH), 123.2, 124.5, 124.6, 130.7,
140.0, 150.0 (Ar). Elemental anal. Found: C, 81.09; H, 9.07. Calcd.
for C34H46O3: C, 81.23; H, 9.22.
Inspired by trinuclear Zn sites in enzymatic systems, a C3-
symmetric trinuclear Zn(II) hydroxide complex of 5 was synthe-
sized and fully characterized using NMR spectroscopy and X-ray
crystallography. This complex at 5 mM concentration induces a
16 900-fold rate enhancement in the catalytic cyclization of the
RNA model substrate, 2-hydroxypropyl-p-nitrophenyl phosphate
(HPNP, pH 6.7, 25 ◦C), over the uncatalyzed reaction with
multiple catalyst turnovers.
1
On the time scale of the H NMR spectra, a C3-symmetric
core is maintained in solution under the conditions used for the
kinetic experiments (pH 6.7, 50% (v/v) CD3CN–D2O, buffer, [7] =
7.5 mM)but theremay be various trinuclear zinc species insolution
at and around pH 6.7 as depicted by the species distribution
diagram. The proton exchange reaction between the various
species appears to be sufficiently fast in 50% (v/v) CD3CN–D2O
on the NMR time scale and thus some sort of hydroxide bridged
C3-symmetric species was observed.
Tris(2-ethylacetoxy-3-methyl-5-tert-butylphenyl)methane (2)
Following
a previously published method for the prepa-
ration of tris(3,5-di-tert-butyl-2-ethoxycarbonylmethoxyphenyl)
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methane,54,81 in a Schlenk flask under argon 0.56 g (1.1 mmol)
of 1 was dissolved in dry acetone and 0.61 g (3.7 mmol) of
ethyl bromoacetate and 1.43 g (4.4 mmol) of Cs2CO3 was added.
The mixture was refluxed for 12–15 h and then cooled to room
temperature. The acetone was removed under vacuum and the
solids dissolved in diethyl ether. Solid MgSO4 was added and the
insoluble salts and drying agent were filtered off. The ether was
removed from the filtrate to give the crude product. The white
solid was recrystallized in ethanol to give 0.72 g (85%) of product.
H, CH), 6.83 (d, 4J(H,H) = 2.4 Hz, 3 H, Ar–H), 6.95 (d, 4J(H,H) =
2.4 Hz, 3 H, Ar–H), 7.32 (d, 3J(H,H) = 8.1 Hz, 6 H, SO2Ar–H),
3
7.78 (d, J(H,H) = 8.1 Hz, 6 H, SO2Ar–H). 13C NMR (CDCl3):
d = 16.5 (Ar–CH3), 21.6 (SO2Ar–CH3), 31.3 (Ar–C(CH3)3), 34.1
(Ar–C(CH3)3), 36.8 (C–H), 69.5 (Ar–O–CH2CH2), 69.6 (Ar–O–
CH2CH2), 125.4, 126.2, 127.9, 129.9, 130.1, 133.0, 135.8, 144.7,
146.1, 151.9 (Ar). Elemental anal. Found: C, 66.77; H, 6.96. Calcd
for C61H76O12S3: C, 66.76; H, 6.98.
−1
1
=
IR: m [cm ] 1725 (C O). H NMR (CDCl3): d = 1.14 (s, 27 H,
3
Preparation of tris[2-{2-{4-[(2-pyridyl)methyl]piperazine}-1-
ethoxy}-3-methyl-5-tert-butylphenyl]methane (5)
Ar–C(CH3)3), 1.30 (t, J(H,H) = 7.1 Hz, 9 H, COOCH2CH3),
2.23 (s, 9 H, Ar–CH3), 4.16 (s, 6 H, Ar–O–CH2–COOEt), 4.23 (q,
3J(H,H) = 7.1 Hz, 6 H, COOCH2CH3), 6.48 (s, 1 H, CH), 6.72 (d,
4J(H,H) = 2.4 Hz, 3 H, Ar–H), 6.99 (d, 4J(H,H) = 2.4 Hz, 3 H, Ar–
H). 13C NMR (CDCl3): d = 14.1 (OCH2CH3), 16.6 (Ar–CH3), 31.3
(Ar–C(CH3)3), 34.1 (Ar–C(CH3)3), 38.1 (C–H), 61.0 (OCH2CH3),
69.5 (Ar–O–CH2–COOEt), 125.7, 126.4, 129.8, 135.4, 146.1, 152.4
To 0.74 g (0.61 mmol) of 4 dissolved in 50 mL dry acetonitrile was
added 3.2 g (18.2 mmol) of 1-[(2-pyridyl)methyl]piperazine, and
0.64 g (6.1 mmol) of Na2CO3, and the mixture was refluxed for 5 d
under an inert atmosphere. The solvent was then removed under
vacuum and the remaining material dissolved in diethyl ether. The
organic layer was extracted with 0.1 M NaOH (2 × 100 mL), then
dried with Na2SO4, filtered, and the solvent removed to afford
0.62 g (91%) of the brownish–yellow amorphous solid. The excess
1-[(2-pyridyl)methyl]piperazine was recovered from the aqueous
phase by extraction with chloroform and removing the solvent
under vacuum. Slow diffusion of pentane into a concentrated
tetrahydrofuran solution of ligand at −30 ◦C afforded crystals
suitable for X-ray analysis. 1H NMR (CD3CN): d = 1.12 (s, 27 H,
Ar–C(CH3)3), 2.21 (s, 9 H, Ar–CH3), 2.39 (b, 24 H, N–CH2CH2–
=
(Ar), 169.2 (C O). Elemental anal. Found: C, 72.49; H, 8.54.
Calcd. for C46H64O9: C, 72.60; H, 8.48.
Tris[2-(2-hydroxylethoxy)-3-methyl-5-tert-
butylphenyl]methane (3)
A dry diethyl ether solution of 2 (1.47 g, 1.9 mmol) was added
dropwise over 1–2 h with an addition funnel to a slurry of LiAlH4
(0.44 g, 11.7 mmol) in 100 mL dry diethyl ether cooled to 0◦
C. The mixture was then warmed to room temperature and stirred
12–15 h. The excess reductant was destroyed with 1 M HCl
(100 mL). The ether layer was separated and further extracted with
1 M HCl (2 × 100 mL) and brine (100 mL). The ether was then
dried with MgSO4. After filtration of the drying agent, the ether
was removed to give 1.06 g (88%) of white crystalline material.
3
N), 2.57 (t, J(H,H) = 6.3 Hz, 6 H, Ar–O–CH2CH2N), 3.52 (t,
3J(H,H) = 6.3 Hz, 6 H, Ar–O–CH2), 3.56 (s, 6 H, Py–CH2), 6.52
4
(s, 1 H, CH), 6.78 (d, J(H,H) = 2.4 Hz, 3 H, Ar–H), 7.07 (d,
3
4J(H,H) = 2.1 Hz, 3 H, Ar–H), 7.17 (ddd, J(H,H) = 7.7 Hz,
5
4J(H,H) = 5.0 Hz, J(H,H) = 1.2 Hz, 3 H, Py), 7.40 (m, 3 H,
Py), 7.68 (dt, 3J(H,H) = 7.7 Hz, 4J(H,H) = 2.0 Hz, 3 H, Py), 8.47
(ddd, 3J(H,H) = 5.4 Hz, 4J(H,H) = 1.7 Hz, 5J(H,H) = 0.9 Hz, 3 H,
Py). 13C NMR (CD3CN): d = 17.1 (Ar–CH3), 31.8 (Ar–C(CH3)3),
34.8 (Ar–C(CH3)3), 38.8 (C–H), 54.2 (Ar–O–CH2CH2–N–CH2),
54.6 (CH2–N–CH2–Py), 58.7 (Ar–O–CH2CH2), 65.2 (N–CH2–
Py), 71.0 (Ar–O–CH2), 123.0 (Py), 123.8 (Py), 126.5 (Ar), 127.0
(Ar), 131.2 (Ar), 137.3 (Ar), 137.5 (Py), 146.4 (Ar), 149.9 (Py),
154.2 (Ar), 160.0 (Py). Elemental anal. Found: C, 73.14; H, 9.16;
N, 10.72. Calcd. for 5·2H2O (C70H101N9O8): C, 73.20; H, 8.86; N,
10.98. ESI FT-ICR MS m/z = 1112.78 [M + H]+.
1
IR: m [cm−1] 3454 (OH). H NMR (CDCl3): d = 1.18 (s, 27 H,
Ar–C(CH3)3), 2.25 (s, 9 H, Ar–CH3), 3.79 (t, 3J(H,H) = 7.8 Hz,
6 H, Ar–O–CH2), 3.84 (m, 6 H, Ar–O–CH2CH2OH), 4.06 (t,
3J(H,H) = 5.5 Hz, 3 H, CH2CH2OH), 6.79 (s, 1 H, CH), 6.92 (d,
4J(H,H) = 2.4 Hz, 3 H, Ar–H), 7.01 (d, 4J(H,H) = 2.4 Hz, 3 H, Ar–
H). 13C NMR (CDCl3): d = 16.9 (Ar–CH3), 31.3 (Ar–C(CH3)3),
34.1 (Ar–C(CH3)3), 36.7 (C–H), 62.0 (Ar–O–CH2CH2OH), 74.0
(Ar–O–CH2CH2OH), 125.5, 126.1, 129.8, 135.8, 145.7, 152.0 (Ar).
Elemental anal. Found: C, 75.50; H, 9.25. Calcd for C40H58O6: C,
75.67; H, 9.21.
Tris[2-(2-toluenesulfonlyethoxy)-3-methyl-5-tert-
butylphenyl]methane (4)
Preparation of hydrochloride salt of tris[2-{2-{4-[(2-
pyridyl)methyl]piperazine}-1-ethoxy}-3-methyl-5-tert-
butylphenyl]methane (6)
In a dry flask 3.83 g (6 mmol) of 3 was dissolved in 100 mL of dry
◦
pyridine and cooled to 0 C in an ice bath. A 4.58 g (24 mmol)
The HCl salt of of 5 (tris[2-{2-{4-[(2-pyridyl)methyl]piperazine}-
1-ethoxy}-3-methyl-5-tert-butylphenyl]methane) was prepared by
dissolving it in diethyl ether and adding 2 N HCl in diethyl
ether resulting in the formation of a white precipitate, which is
thoroughly washed with diethyl ether and dried under vacuum.
portion of toluene sulfonyl chloride was added and the reaction
mixture was stirred for 2 h at 0 ◦C and then for 12–15 h at room
temperature. The pyridine was removed under vacuum and the
solid material dissolved in 100 mL methylene chloride and then
extracted with 1 M HCl (2 × 100 mL). The organic phase was then
dried with MgSO4, filtered, and the solvent removed. Methanol
was added and the white solid product filtered off and washed
with more dry methanol to afford 5.35 g (74%) product. 1H NMR
(CDCl3): d = 1.14 (s, 27 H, Ar–C(CH3)3), 2.13 (s, 9 H, Ar–CH3),
2.42 (s, 3 H, SO2Ar–CH3), 3.49 (t, 6 H, 3J(H,H) = 6.0 Hz, Ar–O–
CH2), 4.14 (t, 6 H, 3J(H,H) = 6.0 Hz, Ar–O–CH2CH2), 6.45 (s, 1
1
The product formed was 5·9HCl·4H2O·Et2O. H NMR (D2O):
d = 1.02 (s, 27 H, Ar–C(CH3)3), 2.31 (s, 9 H, Ar–CH3), 3.05–4.19
(m, 42 H), 6.60 (s, 1 H, CH), 6.64 (s, 3 H, Ar–H), 7.21 (s, 3 H,
Ar–H), 8.04 (m, 6 H, Py), 8.57 (dt, 3J(H,H) = 7.95 Hz, 4J(H,H) =
1.5 Hz, 3 H, Py), 8.77 (d, 4J(H,H) = 5.4 Hz, 3 H, Py). 13C NMR
(D2O): d = 16.87 (Ar–CH3), 31.19 (Ar–C(CH3)3), 34.07 (Ar–
C(CH3)3), 49.11 (C–H), 50.85 (Ar–O–CH2–CH2–N–CH2), 52.04
3932 | Dalton Trans., 2007, 3924–3935
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Table 6 Crystal dataa and structure refinement for 5 and 7
(CH2–N–CH2–Py), 56.07 (Ar–O–CH2–CH2), 57.1 (N–CH2–Py),
65.52 (Ar–O–CH2), 125.61 (Py), 126.49 (Py), 127.23 (Ar), 127.58
(Ar), 128.44 (Ar), 130.87 (Ar), 135.98 (Py), 141.58 (Ar), 147.2
(Py), 152.1 (Ar), 152.37 (Py). Elemental anal. Found: C, 55.75; H,
8.13; N, 8.29. Calcd. for 5·9HCl·4H2O·Et2O (C74H124Cl9N9O8): C,
56.01; H, 7.88; N, 7.94. The presence of nine HCl molecules per
molecule of the ligand is further supported by the observation that
nine equivalents of KOH are required to completely neutralize one
equivalent of the protonated ligand, 6, as depicted in Fig. 5 (vide
supra).
5·C4H8O
7·3(CH3)2CO
Formulaa
C74H105N9O4
1184.67
R3c
16.7988(5)
16.7988(5)
102.377(4)
25020.1(15)
12
C79H109Cl3N9O21Zn3
1823.21
P3
16.7532(16)
16.7532(16)
23.629(3)
5743.4(11)
2
Fw/g mol−1a
Space group
¯
¯
˚
a/A
˚
b/A
˚
c/A
3
˚
V/A
Z
l/mm−1
0.059
0.0805
0.2202
0.747
0.0849
0.2355
b
R1
c
wR2
Preparation of [5·Zn3(l-OH)3](ClO4)3 (7)
a
˚
Obtained with monochromatic Mo-K radiation (k = 0.71073 A) at 173 K.
(Caution! Although, the perchlorate salt is moderately stable, it is
a potential hazard and should therefore be handled with care.) 5
(20 mg, 0.018 mmol) was dissolved in 1 mL acetone and added to
a solution of Zn(ClO4)2·6H2O (20.11 mg, 0.054 mmol) in 1 mL
of acetone mixed with a few drops of methanol (to dissolve
Zn(ClO4)2·6H2O completely). Diffusion of pentane at 0◦ C into
the above solution afforded colorless crystals suitable for X-ray
b R1 = R(ꢁF0| − |Fcꢁ) / R|F0|. c wR2 = [R[w(F0 − Fc2)2] / R[w(F02)2]]1/2
,
2
w = 1/[r2(F02) + [(ap)2 + bp], where p = [F0 + 2Fc2]/3.
2
Potentiometric titrations
Potentiometric titrations were conducted in 50% (v/v) CH3CN–
H2O mixture at an ionic strength of 0.1 M KCl with a Metrohm
702SM titrator equipped with a Metrohm combined pH glass
electrode. The electrode system was calibrated before each mea-
surement by titrating a known amount of HCl in the solvent
mixture with a known concentration of KOH. The base solution
with a concentration of ca. 0.1 M was made in the above solvent
mixture and standardized using potassium hydrogen phthalate.83
A plot of millivolts (measured) vs. pH (calculated) gave a working
slope and intercept so that pH could be read as −log[H+] directly.
The pKw value in 50% (v/v) CH3CN–H2O was determined to
be 15.19 at 25 ◦C and is in good agreement with the published
value.84 The electrode was stored in solvent mixture between
measurements.
All solutions were prepared with purified CH3CN and freshly
boiled 18 MX Millipore deionized water cooled in a nitrogen
stream. All solutions were carefully protected from air by a stream
of nitrogen gas. The linearity of electrode response and carbonate
contamination of the standardized KOH solution was determined
by Gran’s method and was found to be less than 2%.63,64,85,86 The
stock solution of Zn(II) (0.02 M) was prepared by dissolving
Zn(ClO4)2·6H2O in 50% (v/v) CH3CN–H2O. This material was
standardized with EDTA using Eriochrome Black T as indicator.
The potentiometric titration of 6 (0.92 mM) in the absence and
presence of 1, 2 and 3 equivalents of Zn(II) respectively were
carried out at 25 ◦C with I = 0.1 M (KCl) under a nitrogen
atmosphere. The equilibrium constants were calculated using the
program BEST.63 All r fit values (as defined in the program) were
smaller than 0.025. Species distribution was calculated using the
program SPE.63 At least two titration experiments (each with
approximately 100 data points) were performed. The stepwise
constants are denoted by K while the overall constants are denoted
by b.
1
analysis. Isolated yield (17.9 mg, 60%). H NMR (CD3CN): d =
1.20 (s, 27 H, Ar–C(CH3)3), 2.20 (s, 3 H, O–H), 2.29 (s, 9 H, Ar–
CH3), 2.6–3.7 (b, 36 H, N–CH2–CH2–N & Ar–O–CH2CH2N),
4
4.17 (s, 6 H, Py–CH2), 7.10 (s, 1 H, CH), 7.12 (d, J(H,H) =
2.4 Hz, 3 H, Ar–H), 7.20 (d, 4J(H,H) = 2.4 Hz, 3 H, Ar–H), 7.70
(m, 6 H, Py), 8.20 (dt, 3J(H,H) = 7.7 Hz, 4J(H,H) = 1.8 Hz, 3 H,
3
Py), 8.80 (d, J(H,H) = 5.1 Hz, 3 H, Py). 13C NMR (CD3CN):
d = 16.8 (Ar–CH3), 31.7 (Ar–C(CH3)3), 34.9 (Ar–C(CH3)3),
35.6 (C–H), 55.6 (Ar–O–CH2–CH2–N–CH2), 55.8 (CH2–N–CH2–
Py), 58.3 (Ar–O–CH2–CH2), 63.0 (N–CH2–Py), 74.8 (Ar–O–
CH2), 125.9 (Ar), 126.2 (Py), 126.9 (Ar), 131.5 (Ar), 137.2
(Ar), 143.2 (Py), 147.1 (Ar), 150.5 (Py), 153.4 (Ar), 155.1 (Py).
Elemental anal. Found: C, 49.70; H, 5.99; N, 7.42; Cl, 6.24. Calcd.
for 7·2H2O (C70H104N9O20Cl3Zn3): C, 49.63; H, 6.19; N, 7.44;
Cl, 6.28.
X-Ray crystallography†
Unit cell dimensions and intensity data for all the structures were
obtained on a Siemens CCD SMART diffractometer at 173 K. The
data collections nominally covered a hemisphere of reciprocal
space, by a combination of three sets of exposures; each set had a
different φ angle for the crystal, and each exposure covered 0.3◦ in
x. The crystal to detector distance was 5.0 cm. The data sets were
corrected for absorption using SADABS.82
All the structures were solved using the Bruker SHELXTL
software package for the PC, using the direct methods option
of SHELXS. The space groups for the structures were determined
from an examination of the systematic absences in the data, and
the successful solution and refinement of the structures confirmed
these assignments. All hydrogen atoms were assigned idealized
locations and were given a thermal parameter equivalent to 1.2
to 1.5 times the thermal parameter of the atom to which it was
attached. For the methyl groups, where the location of hydrogen
atoms was uncertain, the AFIX 137 card was used to allow the
hydrogen atoms to rotate to the maximum area of residual density,
while fixing their geometry. Structural and refinement data for 5
and 7 are presented in Table 6.
Kinetic measurements
UV-Vis spectra and kinetic traces were recorded with a diode
array spectrometer equipped with a thermostated multicell cuvette
holder (7 cuvettes, 1.0 cm path length) (Hewlett Packard 8453).
Solutions for kinetic measurements were made by adding CH3CN
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(spectrophotometric grade) upto 50% (v/v) to a 80 mM aqueous
buffer solution adjusted with NaOH to the desired pH. Buffers
(MES, pH 5.6–7.0 and HEPES, pH 7.0–8.2) were obtained from
commercial sources and used without further purification in
18 MX Millipore deionized water. Stock solutions were freshly
prepared before performing the kinetic measurements. In a typical
experiment, preformed complex 7 (1040 lL, 10 mM in CH3CN)
was added to a cuvette containing 38 lL of CH3CN and 962.4 lL
of 173.25 mM aqueous buffer solution and thermostated at
25 ◦C. After a couple of minutes of equilibration time, HPNP
(41.6 lL, 100 mM in water) was injected and an increase in
the UV absorption at k = 400 nm due to the release of p-
nitrophenolate ion was recorded. All solutions remained clear
during the time of the kinetic measurements. In the absence of
ligand, precipitation of polymeric Zn(II) hydroxide took place.
The observed pseudo-first-order rate constants kobs (s−1) were
calculated with the extinction coefficient of p-nitrophenolate at
k = 400 nm by the initial slope method (<5% conversion).
The method of initial rates was used to study these reactions
as the catalyst was not stable for even one complete half-life
of the reaction in the presence of excess buffer. Solutions of p-
nitrophenolate in CH3CN–20 mM buffer 1 : 1 (v/v) were prepared
at various pH values. The molar extinction coefficient for p-
nitrophenolate at 400 nm was then determined from the plot of
absorbance against concentration. Correction for the spontaneous
hydrolysis of HPNP (less than 0.1%) was accomplished by direct
observation of the production of p-nitrophenolate relative to a
reference cell containing no metal complex. The pseudo-first-order
rate constants for the transesterification of HPNP in the absence
of the catalyst (kuncat/s−1) were measured with a 2.0 mM HPNP
solution by the method of initial rates. Each experiment was run
in triplicate. Agreement between the calculated initial rates for
replicate experiments was within 5%.
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Acknowledgements
We thank the National Science Foundation (Grant CHE-0316003)
for funding this research. We are especially grateful to Professor
David E. Richardson and Dr Kathryn R. Williams at the
University Of Florida for the use of instruments and for helpful
discussions. We also thank Daniel E. Denevan for his help with
the UV experiments.
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