Solvent induced cooperativity of Zn(ii) complexes cleaving a phosphate diester RNA analog in methanol

Article abstract of DOI:10.1039/c1ob06482g

The kinetics of cyclization of 2-hydroxypropyl p-nitrophenyl phosphate (1) promoted by two mononuclear Zn(ii) catalytic complexes of bis(2-pyridylmethyl) benzylamine (4) and bis(2-methyl 6-pyridylmethyl)benzylamine (5) in methanol were studied under sspH-controlled conditions (where sspH refers to [H ⁺] activity in methanol). Potentiometric titrations of the ligands in the absence and presence of Zn²⁺ and a non-reactive model for 1 (2-hydroxylpropyl isopropyl phosphate (HPIPP, 6)) indicate that the phosphate is bound tightly to the 4:Zn(ii) and 5:Zn(ii) complexes as L:Zn(ii):6^-, and that each of these undergoes an additional ionization to produce L:Zn(ii):6^-:(^-OCH₃) or a bound deprotonated form of the phosphate, L:Zn(ii):6^2-. Kinetic studies as a function of [L:Zn(ii)] indicate that the rate is linear in [L:Zn(ii)] at concentrations well above those required for complete binding of the substrate. Plots of the second order rate constants (defined as the gradient of the rate constant vs. [complex] plot) vs.sspH in methanol are bell-shaped with rate maxima of 23 dm mol^-1 s^-1 and 146 dm mol^-1 s^-1 for 4:Zn(ii) and 5:Zn(ii), respectively, at their sspH maxima of 10.5 and 10. A mechanism is proposed that involves binding of one molecule of complex to the phosphate to yield a poorly reactive 1:1 complex, which associates with a second molecule of complex to produce a transient cooperative 2:1 complex within which the cyclization of 1 is rapid. The observations support an effect of the reduced polarity solvent that encourages the cooperative association of phosphate and two independent mononuclear complexes to give a reactive entity.

Full text of DOI:10.1039/c1ob06482g

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PAPER
Solvent induced cooperativity of Zn(II) complexes cleaving a phosphate diester
RNA analog in methanol†
Mark F. Mohamed, Irma Sa´nchez-Lombardo, Alexei A. Neverov and R. Stan Brown*
Received 29th August 2011, Accepted 14th October 2011
DOI: 10.1039/c1ob06482g
The kinetics of cyclization of 2-hydroxypropyl p-nitrophenyl phosphate (1) promoted by two
mononuclear Zn(II) catalytic complexes of bis(2-pyridylmethyl)benzylamine (4) and bis(2-methyl
6-pyridylmethyl)benzylamine (5) in methanol were studied under sspH-controlled conditions (where sspH
refers to [H+] activity in methanol). Potentiometric titrations of the ligands in the absence and presence
of Zn2+ and a non-reactive model for 1 (2-hydroxylpropyl isopropyl phosphate (HPIPP, 6)) indicate that
the phosphate is bound tightly to the 4:Zn(II) and 5:Zn(II) complexes as L:Zn(II):6-, and that each of
these undergoes an additional ionization to produce L:Zn(II):6-:(-OCH3) or a bound deprotonated
form of the phosphate, L:Zn(II):62-. Kinetic studies as a function of [L:Zn(II)] indicate that the rate is
linear in [L:Zn(II)] at concentrations well above those required for complete binding of the substrate.
Plots of the second order rate constants (defined as the gradient of the rate constant vs. [complex] plot)
vs. sspH in methanol are bell-shaped with rate maxima of 23 dm mol-1 s-1 and 146 dm mol-1 s-1 for
4:Zn(II) and 5:Zn(II), respectively, at their sspH maxima of 10.5 and 10. A mechanism is proposed that
involves binding of one molecule of complex to the phosphate to yield a poorly reactive 1 : 1 complex,
which associates with a second molecule of complex to produce a transient cooperative 2 : 1 complex
within which the cyclization of 1 is rapid. The observations support an effect of the reduced polarity
solvent that encourages the cooperative association of phosphate and two independent mononuclear
complexes to give a reactive entity.
of the RNA model 2-hydroxypropyl-p-nitrophenyl phosphate
Introduction
(HPNPP, 1) vs. [2:Zn(II):(-OCH3)] in methanol exhibited upward
The enzymes responsible for the cleavage of the phosphate diester
curvature consistent with a process which is second order in
-
linkage (ROP(O2 )–OR¢) in DNA and RNA are among the most
efficient known, achieving accelerations for P–OR¢ hydrolysis
or transesterification of up to 1017 times.1 Many such phos-
phodiesterase enzymes contain two or more metal ions in their
active sites2 which has spurred imaginative approaches to develop
[2:Zn(II)]. This suggested that a corresponding dinuclear complex
such as 3:Zn(II)2 should have even greater activity, which proved
obs
to be the case, since the k2 for its catalytic cleavage of 1 was
2.75 ¥ 105 dm3 mol-1 s-1: this corresponds to an acceleration of
~108 over the methoxide-promoted reaction in methanol where
-
-
= 2.56 ¥ 10-3 dm3 mol-1 s-1.5a It appears that the switch
MeO
anthropogenic metallo-catalysts that are capable of ROP(O2 )–
k2
OR¢ cleavage with enzyme-like efficiency.3 Based on the notion that
the effective dielectric constants of enzyme active sites resemble
those of organic solvents rather than water,4 we have initiated a
program to investigate the ability of metal containing complexes
to catalyze the reactions of phosphate diesters and triesters in the
light alcohols.5 These studies show that highly active dinuclear
complexes can be generated in the light alcohols, methanol and
ethanol, where they exhibit high levels of metal ion cooperativity
for phosphate diester cleavage.6 In an initial study,7 we found that
plots of the kobs values for catalyzed intramolecular cyclization
from water to a lower dielectric constant alcohol solvent provides
at least three important effects that greatly accelerate Mx+ :(-OR)-
catalyzed phosphoryl transfer reactions. These are: (a) increasing
the electrostatic ion–ion and ion–dipole association of the metal
ion and oppositely charged or polarized substrates; (b) increasing
the solubility of metal ions at ‘pH’ values above the ‘pKa’ of
the Mx+ (HOR)n ꢀ Mx+ (-OR)(HOR)m + H+ ionization; and, (c)
providing a medium effect that accelerates the reactions where
charge is dispersed in transition states.
The results in methanol are in stark contrast to the reported
situation in water, the medium in which most of the work
concerning synthetic metallo-nucleases has been conducted.8 We
have reviewed the problems associated with the study of metallo-
complexes in water.9 Under largely aqueous conditions, dinuclear
complexes, including 3:Zn(II)2,10 characteristically do not show
any clear sign of metal ion cooperativity toward the cleavage
Department of Chemistry, Queen’s University Kingston, Ontario, Canada,
K7L 3N6. E-mail: rsbrown@chem.queensu.ca; Fax: +1-613-533-6669;
Tel: +1-613-533-2400
† Electronic supplementary information (ESI) available: Tables and Fig-
ures giving various kobs vs. [catalyst] rate constant data for cleavage of 1 as
well as the 1H NMR spectrum of 4. See DOI: 10.1039/c1ob06482g
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of phosphate esters, and in many cases are not better than
the hydroxide-promoted reaction in terms of second order rate
constant. Albeit rare, there are standout cases8c,g,11 where dinuclear
complexes are over two orders of magnitude more reactive toward
the cyclization of 1, or cleavage of other phosphate diesters in
water, than are the appropriate mononuclear comparison and the
-OH-promoted reaction.
To our knowledge, there are no reports of studies describing
the aqueous reactivity of mononuclear complexes that promote
the cleavage of phosphate esters where the kinetics are greater
than first order in [complex].12 The plot of kobs vs. [2:Zn(II):L:-OH]
for the catalyzed cleavage of HPNPP in water is strictly linear,
signifying no appreciable substrate binding nor kinetic terms
higher than first order in catalyst.12e However, Deal and Burstyn13
observed saturation kinetics for the hydrolysis of O-ethyl O-4-
nitrophenyl phosphate promoted by the Cu(II) complex of 1,4,7-
triazacyclononane, indicative of the formation of a kinetically
active 1 : 1 catalyst:substrate complex with relatively weak binding
(KM = 0.062 mol dm-3) with no evidence for a higher than first
order dependence on [catalyst].
Experimental
(a) Materials
Methanol (DriSolv) was purchased from EMD Chemicals.
Zn(OTf)2 (98%), sodium methoxide (0.50 M solution in methanol,
titrated against N/50 certified standard aqueous HCl solution
and found to be 0.49 M), 2,6-lutidine (98%), N-methylpiperidine
(99%), 4-ethylmorpholine (98%), triethylamine (99%), 2,2,6,6-
tetramethylpiperidine (99%), trifluoromethanesulfonic acid, ben-
zylamine (99%), and 2-picolylchloride hydrochloride (98%) were
purchased from Aldrich and used without further purification.
2-Hydroxypropyl iso-propyl phosphate 6 was available from a
previous study.16
(b) Synthesis
Bis(2-pyridylmethyl)benzylamine (4). Benzylamine (0.66 g,
0.006 mol) and 2-picolylchloride hydrochloride (2.07 g, 0.012 mol)
were dissolved in 15 mL of water and heated to 60 C in an oil
◦
bath. To the stirring mixture was added 7 mL of 5 N aqueous
NaOH drop-wise, after which the mixture was stirred at 60 ◦C
for one hour. The mixture was cooled to room temperature and
then extracted with 3 ¥ 20 mL CH2Cl2. The combined extracts
were dried (Na2SO4) and then evaporated to give a brown oil. The
crude product was purified by flash silica gel chromatography on
a Biotage SP1 purification system, using CHCl3/HOCH3 (10 : 1)
as the eluent (Rf 0.15). The purified product (1.3 g, 73% yield)
was obtained as a yellow oil whose spectral characteristics were
consistent with those previously published.17
1H NMR (400 MHz, CDCl3): shown in the ESI;† d 8.53 (2H, d,
J = 4 Hz), d 7.67 (2H, t, J = 8 Hz), d 7.60 (2H, d, J = 8 Hz), d 7.42
(2H, d, J = 4 Hz), d 7.33 (2H, t, J = 4 Hz), d 7.24 (1H, t, J = 8 Hz),
d 7.15 (2H, t, J = 4 Hz), d 3.82 (4H, s), d 3.70 (2H, s). 13C NMR
(150 MHz, CDCl3): d 58.6, 60.0, 121.9 122.7, 127.1, 128.3, 128.8,
Although we have provided several examples of dinuclear com-
plexes exhibiting strong metal ion cooperativity in methanol,6,14,15
it is an open question whether the low polarity of alcohol solvents
might prove more general in recruitment of two mononuclear
complexes such as 2:Zn(II) for cooperative cleavage of phosphate
diesters.6a,c Herein, we report a kinetic and potentiometric titration
study of the cyclization of 1 promoted by two other complexes in
methanol, 4:Zn(II) and 5:Zn(II), where each system demonstrates
such cooperativity. The present cases also demonstrate: (1) strong
1 : 1 binding between a mononuclear complex and substrate 1; and,
(2) that the medium facilitates a cooperative mechanism involving
a transient 2 : 1 complex:substrate super-complex that promotes
the intramolecular cyclization reaction of 1 at rates approaching
what is achievable by dinuclear complexes where the catalytic units
are covalently linked.
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136.4, 138.9, 148.9, 159.8. HRMS (EI-TOF) calcd for C19H19N3
electrode calibrated with Fischer certified standard aqueous
buffers (pH 4.00 and 10.00) as described.6,14,15 Potentiometric
titrations of 4, 5, Zn(OTf)2 and the sodium salt of 2-hydroxylpropyl
iso-propyl phosphate (HPIPP, 6:Na) were performed under Ar
in methanol at 25 ◦C. For the metal ion/ligand and metal
ion/ligand/phosphate titration experiments the concentration of
metal ion, ligand and phosphate were all 1 ¥ 10-3 mol dm-3, and
the final concentration of added acid, HOTf, was 4 ¥ 10-3 mol
dm-3. The total sample volume in all cases was 20.0 mL. Sodium
methoxide titrant was calibrated by titrating Fischer certified
standard HCl in water, with the endpoint taken to be wwpH 7.
The potentiometric data were analyzed using the computer pro-
gram Hyperquad 2000 (version 2.1 NT),21 with the autoprotolysis
constant of pure methanol taken to be 10-16.77 (mol dm-3)2 at 25 ◦C.
The dissociation constants for the species 4-H+n or 5-H+n (n = 1–3)
were determined from the analysis of the potentiometric titration
data at 1 ¥ 10-3 mol dm-3 4 or 5. Titrations were performed in
duplicate and were analyzed separately. The respective species
formation constants were averaged and the resulting values were
used as constants in the subsequent Hyperquad analysis of 1 : 1
4:Zn(II) or 5:Zn(II) titrations. Similarly, formation constants for
L:Zn(II), L(-OCH3):Zn(II) and Zn(II):(-OCH3)2 (L = 4,5) were
determined from the analysis of the potentiometric titrations of
1 ¥ 10-3 mol dm-3 Zn(OTf)2 with an equimolar amount of 4 or 5,
and the resulting values were used as constants in the subsequent
Hyperquad analysis of 1 : 1 : 1 L:Zn(II):Phos titrations.
(M+): 289.1579; found 289.1592. lmax(HOCH3) = 260 nm.
Bis(2-methyl 6-pyridylmethyl)benzylamine (5). To a suspen-
sion of 2-bromomethyl-6-methylpyridine18 (2.29 g, 0.012 mol) in
15 mL of water was added benzylamine (0.64 mL, 0.0059 mol) and
the mixture was heated with stirring to 60 ◦C. To this was added
10 mL of 5 N aqueous NaOH drop-wise, after which the mixture
was stirred at 60 ◦C for one hour during which the mixture became
clear. This was cooled to room temperature and then extracted
with 2 ¥ 20 mL CH2Cl2. The combined extracts were dried
(Na2SO4) and then evaporated to give a viscous orange oil. The
crude product was purified by flash silica gel chromatography on
a Biotage SP1 purification system, using CHCl3/HOCH3 (40 : 1)
as the eluent (Rf 0.15). The purified product (1.2 g, 65% yield) was
obtained as a pale yellow oil which formed an off-white solid upon
◦
standing. Mp = 76–77 ◦C (lit. 76.9 C19). H NMR (300 MHz,
1
CDCl3): d 7.57 (2H, t, J = 9 Hz), d 7.45 (4H, dd, J = 9 Hz), d
7.30 (3H, m), d 7.00 (2H, d, J = 9 Hz), d 3.80 (4H, s), d 3.70
(2H, s), d 2.53 (6H, s). 13C NMR (150 MHz, CDCl3): d 24.8,
58.9, 60.5, 119.7, 121.7, 127.3, 128.6, 129.2, 137.0, 139.6, 157.8,
159.8. HRMS (EI-TOF) calcd for C21H23N3 (M+): 317.1892; found
317.1878. Anal. Calcd for C21H23N3: C, 79.46; H, 7.30; N, 13.24.
Found: C, 79.65; H, 7.20; N, 13.23. lmax(HOCH3) = 270 nm.
(c) Kinetics
Kinetics in methanol using UV-Visible spectroscopy. The kinet-
ics of catalyzed cleavage of 1 (5.0 ¥ 10-5 mol dm-3 in methanol)
were monitored by UV-vis spectrophotometry at 25.0 0.1 ◦C by
observing the rate of appearance of p-nitrophenol at 320 nm or
p-nitrophenolate at 400 nm. First order rate constants (kobs) were
obtained from fitting the absorbance vs. time traces to a standard
exponential model. All kinetic experiments were performed in
quartz cuvettes with catalyst formed in situ through sequential
addition of stock solutions (typically 1.0 ¥ 10-1 mol dm-3) of
each of ligand and Zn(OTf)2 to buffered methanol solutions to
make a total volume of 2.5 mL. Reactions were initiated by
the addition of substrate to this solution. Buffer solutions were
prepared at 20.0 mmol dm-3 using the following amines and triflic
Results
(i) Potentiometric titration and speciation analysis
Given in Table 1 are the various constants determined from the
potentiometric titration of the L = 4 and 5 systems which were
obtained as follows. For simplicity throughout, the various forms
are depicted as L:Zn(II) when the Zn2+ is simply bound to the
ligand, and L:Zn(-OCH3), or L:Zn:Phos when the Zn2+ is bound
to methoxide or to the anionic form of phosphate esters 6- or
1-. Independent titrations of 1.0 mol dm-3 solutions of each
s
acid (HOTf) in methanol to adjust the pH20 of the solution
Table 1 Formation constants and acid dissociation constants for various
species determined by potentiometric titration
s
s
s
s
s
(2,6-lutidine, pH = 7.60; N-ethylmorpholine, pH = 8.00–9.10;
N-methylpiperidine, sspH = 9.90; triethylamine, sspH = 10.10–11.3;
2,2,6,6-tetramethylpiperidine, pH = 12.00–12.10). Where buffer
Log ssKa
Log ssKa
s
s
Equilibrium
L = 4 system L = 5 system
inhibition was observed, plots of kobs vs. [buffer]total were linear with
a downward slope. Extrapolation of the plot of kobs vs. [buffer]total
to zero concentration gave a buffer-independent rate constant that
was used to correct the original experimental data (see the ESI†).
The reported values of the kobs constants for the production of
p-nitrophenol (phenolate) are the averages of duplicate runs.
[L-H+]↔[L][H+]
-7.15 0.02
7.0 0.1
14.01 0.02
-9.76 0.1
-4.4 0.3
11.5 0.3
18.07 0.1
-7.72 0.02
4.30 0.05
10.97 0.03
-10.10 0.06
-4.4 0.3
[L][Zn]↔ [L:Zn]
[L][-OCH3][Zn]↔[L:Zn(-OCH3)]b
[L:Zn(HOCH3)]↔[L:Zn(-OCH3)][H+]c
[6-H]↔[6-][H+]
[L][Zn][6-]↔[L:Zn:6-]
9.00 0.02
14.77 0.01
+
[L][Zn][-OCH3][6-]↔[L:Zn(-OCH3):6-]d
The [CH3OH2 ] was determined potentiometrically using a
combination glass electrode (Radiometer model no. XC100-111-
a Derived from fits of the potentiometric titration data using the program
Hyperquad 2000. Log ssK refers to the log of the acid dissociation
constant or the log of the equilibrium constant computed in the direction
shown in column 1. b Defined as L + -OCH3 + Zn ꢀ L:Zn(-OCH3),
calculated from -16.77 + b values where -16.77 comes from the auto-
120-161) calibrated with certified standard aqueous buffers (pH =
s
w
4.00 and 10.00). The measured pH meter readings in methanol
were converted to the sspH values by subtracting the d correction
factor of -2.24.20
protolysis constant of methanol of 10-16.77 c Computed from -16.77 +
.
(log([L:Zn(-OCH3)]/[L][-OCH3][Zn]) - log([L:Zn]/[L][Zn]). d Defined as
L + -OCH3 + Zn + 6- ꢀ L:Zn(-OCH3):6-, calculated from -16.77 + b
values where -16.77 comes from the autoprotolysis constant of methanol
(d) Potentiometric titrations
of 10-16.77
.
Titrations were performed in duplicate using an autotitrator
equipped with an Accumet model 13-620-183 combination glass
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ligand were performed, providing the sspKa values for the last acid
dissociation step, of 7.15 0.02 and 7.72 0.02 for 4-H+ and 5-H+,
respectively.
Analysis of the potentiometric titration profile data, as well
as those for an equimolar mixture of Zn(OTf)2 and 4 shown
in Fig. 1, allow us to define the relevant species in solution as
4:Zn(II) and 4:Zn(-OCH3), which, when fit via the Hyperquad
2000 program, provide log b values of 7.00 0.06, and -2.76
0.02. The formation constants for these two species were fixed
for all subsequent analyses of the titration in the presence of
HPIPP (6), a non-reactive structural model of HPNPP (1), and
also for the fitting of the full titration profile for the former one,
which produces a reasonable fit and provides respective stability
constants for 4:Zn:Phos and 4:Zn:(-OCH3):Phos species with log
b values of 11.5 0.3 and 1.3 0.1. In Fig. 2 are titration profiles
for 5 alone, as well as those for an equimolar solution of Zn(OTf)2
with 5 and finally that of 5 with Zn(OTf)2 in the presence of HPIPP.
Hyperquad analysis of the data of the profile of 5 with Zn(OTF)2
and the one for 5 alone, gives log b values of 4.3 0.05 and -5.8
0.03 for the species 5:Zn(II) and 5:Zn (-OCH3).
s
s
Fig. 3 Speciation diagram of various Zn(II) forms as a function of pH
computed from the formation constants (given in the results section)
under kinetic conditions of an equimolar solution of 1 ¥ 10-3 mol dm-3 of
Zn(OTf)2 and 4 in methanol at 25 ◦C.
s
s
Fig. 4 Speciation diagram of various Zn(II) forms as a function of pH
computed from the formation constants (given in the results section)
under kinetic conditions of an equimolar solution of 1 ¥ 10-3 mol dm-3 of
Zn(OTf)2 and 5 in methanol at 25 ◦C.
Fig. 1 Potentiometric titration profiles for 1 ¥ 10-3 mol dm-3 4 and 4 ¥
10-3 mol dm-3 HOTf, (---); the same in the presence of 1 ¥ 10-3 mol dm-3
Zn(OTf)2, ( ◊ ◊ ◊ ); and 1 ¥ 10-3 mol dm-3 4 containing 4 ¥ 10-3 mol dm-3
95% of the total Zn2+ is present as 4:Zn(II) and 4:Zn(-OCH3)
s
s
with the latter having a pKa of 9.76 for its formation. On the
HOTf, 1 ¥ 10-3 mol dm-3 of Zn(OTf)2 and 1 ¥ 10-3 mol dm-3 of HPIPP (
)
other hand, Fig. 4 shows that, with ligand 5, the metal ion is not
bound as tightly, with ~75% existing as 5:Zn(II) at sspH 8.5, and a
maximum of 38% existing as 5:Zn:(-OCH3) at sspH 10.4 (sspKa for
in CH3OH at 25 ◦C.
s
s
its formation 10.1). Above the latter pH, free Zn(II) in solution
and that in 5:Zn(-OCH3) are sequestered further by methoxide to
form Zn(-OCH3)2.
Analysis of the data obtained for the titrations performed in
the presence of HPIPP (6) provides respective stability constants
for L:Zn:6- and L:Zn:(-OCH3):6- or its chemical equivalent where
the 2-hydroxypropyl group is deprotonated (L:Zn:62-). For the
two ligand systems, the respective log b values for formation
of the L:Zn:6- and L:Zn:(-OCH3):6- forms are 11.5 and 1.3 for
ligand 4 and 9.0 and -2.0 for ligand 5. HPIPP (6) is used as a
non-reactive model for the reactive substrate used in the kinetics
(HPNPP (1)) to provide an approximate speciation diagram for the
L:Zn:1- species with the understanding that the different structure
Fig. 2 Potentiometric titration profiles of 1 ¥ 10-3 mol dm-3 5 and 4 ¥
10-3 mol dm-3 HOTf, (---); the same in the presence of 1 ¥ 10-3 mol dm-3
Zn(OTf)2, ( ◊ ◊ ◊ ); and 1 ¥ 10-3 mol dm-3 5 along with 4 ¥ 10-3 mol dm-3
s
and less electron withdrawing leaving group could alter the pH
s
dependence of the various forms. The speciation diagrams for
all the phosphate containing species shown in Fig. 5 and 6 were
constructed using conditions relevant to the kinetics where [4] or
[5] = [Zn(II)] = 1 ¥ 10-3 mol dm-3 and [HPIPP] = 5 ¥ 10-5 mol dm-3.
The concentration of the various [HPIPP] species in the presence
of Zn(II) and 4 or 5 were subsequently computed as a function
of the sspH, inputting the sspKa of HPIPP (4.4 0.3), using HySS
HOTf, 1 ¥ 10-3 mol dm-3 of Zn(OTf)2 and 1 ¥ 10-3 mol dm-3 of HPIPP (
)
in CH3OH at 25 ◦C.
The speciation diagrams for the various forms of the L:Zn
complex, Zn(II), and ligand were constructed using the constants
given in Table 1, and are shown in Fig. 3 and 4. Fig. 3 indicates
that, over the sspH region used for the kinetics studies, more than
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Fig. 5 Speciation diagram of various HPIPP forms as a function of sspH
computed from the formation constants (given in the results section) under
kinetic conditions of HPIPP 5 ¥ 10-5 mol dm-3 in the presence of 1 ¥
10-3 mol dm-3 each of Zn(OTf)2 and 4 in CH3OH at 25 ◦C.
Fig. 7 Plot of kobs vs. [4:Zn(II)] for the cleavage of HPNPP (1) (5 ¥
10-5 mol dm-3) (ꢁ). Data superimposed on the figure as ( ) are
᭹
multiplication products of [4:Zn:6-][4:Zn:(-OCH3)] in accordance with the
bimolecular reaction pathway given in Scheme 1, sspH 10.1 at 25 ◦C. The
cat
average value for the second order rate constant k2 (Scheme 1) was
calculated to be 24 5 dm3 mol-1 s-1. For a discussion of this computed
value and the observed second order rate constant for 4, see below.
Fig. 6 Speciation diagram of various [HPIPP] forms as a function of
sspH computed from the formation constants under kinetic conditions of
HPIPP 5 ¥ 10-5 mol dm-3 in the presence of 1 ¥ 10-3 mol dm-3 each of
Zn(OTf)2 and 5 in CH3OH at 25 ◦C.
Fig. 8 Plot of kobs vs. [5:Zn(II)] for the cleavage of HPNPP (1) (5 ¥ 10-5
mol dm-3) (ꢁ). Data superimposed on the figure as ( ) are multiplication
᭹
2009. One sees, from the speciation diagram in Fig. 5 for ligand
4, that between sspH 4.5 and 8, over 90% of the HPIPP phosphate
diester is bound as 4:Zn:6-, and that above sspH 9.2 the bulk of the
HPIPP exists predominantly 4:Zn(-OCH3):6- or its deprotonated
phosphate form 4:Zn:62-.
products of [5:Zn:6-][5:Zn:(-OCH3)] in accordance with the bimolecular
reaction pathway given in Scheme 1, sspH 9.9 at 25 ◦C. The average value
cat
for the second order rate constant k2 (Scheme 1) was calculated to be
316 50 dm3 mol-1 s-1. For a discussion of this computed value and the
observed second order rate constant for 5, see below.
The speciation diagram in Fig. 4 for ligand 5 and Zn(II) shows
of [L:Zn:6-][L:Zn:(-OCH3)], which explains the upward curvature
in the kobs vs. [L:Zn(II)] kinetic plots shown later.
that, in the absence of phosphate 6, ~75% of the available Zn(II)
s
is bound to the ligand as 5:Zn(II) at pH 7.8, and 50% is bound
s
in that form at sspH 9.8, along with an additional 28% in the form
of 5:Zn:(-OCH3). However, the speciation diagram in Fig. 6 for
(ii) Dependence of the rate constant for cyclization of 1 on
[L:Zn(II)] and sspH
s
s
HPIPP along with 5 and Zn(II) as a function of pH, shows that
more than 90% of the HPIPP is fully bound as 5:Zn:6- between
s
sspH 5.9 and 9.8, and in the higher pH region where the kinetic
The kinetics of the catalyzed cleavage of 1 were studied in
s
s
s
experiments were performed, over 90% of the HPIPP is present as
5:Zn:6- and 5:Zn:(-OCH3):6- (or 5:Zn:62-).
buffered methanol with control of pH and ionic strength. At
s
s
all pH values, plots of kobs vs. [4:Zn(II)] or [5:Zn(II)] exhibited
Fig. 7 and 8 present the observed first order kinetic data (kobs
)
an upward curvature (see Fig. 7 and 8, and the ESI†). This is
consistent with a process that is overall second order in [L:Zn(II)]
or first order in each of [L:Zn(II)] and its ionization product,
[L:Zn:(-OCH3)]. Given in Scheme 1 is the favoured process where
a tightly bound L:Zn:1- complex (predicted on the basis of the
above potentiometric titration data) interacts productively with
a second complex comprising L:Zn:(-OCH3) to promote the
catalytic cyclization of 1. At high catalyst concentrations, higher
than that which ensures formation of a 1 : 1 : 1 L:Zn:1- complex,
the reactions are linear in [catalyst], suggesting the involvement
for the cleavage of HPNPP (1) vs. total [4:Zn(II)] or [5:Zn(II)],
where the right y-axis gives the multiplication product of the
calculated concentrations of [4:Zn:6-][4:Zn:(-OCH3)] at sspH 10.1
s
and [5:Zn:6-][5:Zn:(-OCH3)] at pH 9.9, as determined from the
s
potentiometric titration constants. The calculated concentrations
of the species were computed using conditions where [HPIPP] =
5 ¥ 10-5 mol dm-3 and [4:Zn(II)] or [5:Zn(II)] = 1 ¥ 10-4–2 ¥ 10-3
mol dm-3. Inspection of the plots in Fig. 7 and 8 shows that the
kobs values have excellent correlations with multiplication product
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Scheme 1 L = 4, 5. Minimal scheme for the cyclization of 1 promoted by
two equivalents of L:Zn(II).
cat
Table 2 Computed apparent second order rate constants (k2 app) and
dissociation constants (Kd) for the cyclization of HPNPP (1) catalyzed by
4:Zn(II) at various sspH values
cat
sspH
k2
(dm3 mol-1 s-1)
Kd (mmol dm-3)
app
cat
Fig. 9 Plots of log k2
vs. sspH for the cyclization of 1 (5.0 ¥ 10-5 mol
app
8.0
8.5
9.1
10.1
10.8
11.3
12.1
0.180 0.001
0.850 0.002
2.87 0.01
0.20 0.06
0.17 0.04
0.13 0.03
0.4 0.16
0.1 0.02
—
dm-3) catalyzed by 4:Zn(II) (ꢀ) and 5:Zn(II) (ꢂ) in buffered methanol at
T = 25.0 0.1 ◦C. Lines through the data are obtained from fits to eqn (2).
17
15
3
1
ionizations where the active form of the catalyst is formed after
the first ionization of the complex, and an inactive form formed
after the second ionization). The fitting gives two apparent acid
10.6 0.6
7.7 0.1
—
dissociation constants and a maximum apparent second order rate
cat
cat
max for 4:Zn(II) of sspKa1 = 10.2 0.2; sspK2a = 11.5
Table 3 Computed apparent second order rate constants (k2 app) and
dissociation constants (Kd) for the cyclization of HPNPP (1) catalyzed by
5:Zn(II) at various sspH values
constant (k2
)
app
max
cat
0.15; (k2
)
= 23.1 dm3 mol-1 s-1 and for 5:Zn(II), sspK1a = 8.8
cat max
app
0.1; sspKa2 = 10.6 0.1; (k2
)
= 146.1 dm3 mol-1 s-1
app
cat
sspH
k2
(dm3 mol-1 s-1)
Kd (mmol dm-3)
app
(k2cat
2
)
+
max ssK1a[H+ ]
⎛
⎜
⎜
⎞
⎟
⎟
⎟
⎟
⎟
⎠
app
k2cat
=
(2)
app
⎜
+
s
1
s
1 s
a s
2
a
7.6
8.3
8.6
8.9
10.8 0.9
32.9 1.6
43.6 1.7
79.2 1.4
137.0 2.4
105.2 8.8
43.9 4.6
30.0 1.5
5.6 0.6
1.97 0.20
1.37 0.15
0.64 0.09
1.00 0.05
1.47 0.06
3.09 0.41
1.42 0.34
0.73 0.12
0.93 0.26
⎜
⎜[H ] +[H ] K + K
K
⎝
s
a
s
cat
s
The superpositionings of the data for log k2
vs. pH for the
s
app
cyclization of 1 (5.0 ¥ 10-5 mol dm-3) catalyzed by 4:Zn(II) and
5:Zn(II) in buffered methanol at T = 25.0 0.1 ◦C are shown
in Fig. 39S and 40S of the ESI,† and are seen to correlate well
with the computed concentration profiles as a function of sspH for
the multiplication product of [L:Zn(II):6][L:Zn(-OCH3)]. Bearing
in mind that the potentiometric and kinetic data are obtained
under different conditions (buffered conditions with 1 for kinetics
and non-buffered conditions with 6 for titrations), the Fig. 39S†
concentration plot, constructed considering only the latter two
species as the active forms, fits the ascending data at low sspH well,
9.9
10.4
10.9
11.2
12.0
of a second catalyst molecule in the mechanism for cleavage of
1-. The kinetic data were fit to eqn (1), derived for the process in
Scheme 1, which incorporates a universal binding equation.6,14,15
This gives the L:Zn:1- ꢀ L:Zn(II) + 1- dissociation constant (Kd,
taken as the reciprocal of the binding constant, KB, from eqn (1))
as well as the apparent second order rate constant for the reaction
cat
but undershoots the k2 app vs. sspH data for 4 suggesting that some
other species is active for catalysis at sspH > 10. With 5:Zn(II), the
similar plot shown in Fig. 40S† fits the available kinetic acceptably
well. Strong binding of an alternative anionic phosphate diester
(dibenzyl phosphate) to these complexes is also demonstrated
by an inhibition of the reaction of complex with a non-binding
substrate (O-4-nitrophenyl O-ethyl methylphosphonate) shown in
the ESI (Table 15S, Fig. 15S, and Table 35S, Fig. 35S).†
cat
between the L:Zn:1- complex and free catalyst (k2 app). Note that
in the fit of the data it is not necessary to include the term k1cat for
the unimolecular breakdown of the L:Zn:1- complex, suggesting
that under the present conditions, this is too slow to be of any real
kinetic benefit.
cat
kobs = k2 app([Cat] - (1 + KB ¥ [S] + [Cat] ¥ KB - X)/
(2KB)/[S]) ¥ (1 + KB ¥ [S] + [Cat] ¥ KB - X)/(2KB)/[S])
(1)
where:
(iii) Turnover experiments for the cleavage of 1 catalyzed by
4:Zn(II) and 5:Zn(II)
X = (1+ 2KB ¥ [S] + 2 ¥ [Cat] ¥ KB + KB ¥ [S]2- 2 ¥ KB
¥
2
2
2
0.5
[Cat][S] + [Cat]2 ¥ KB
)
Kinetic experiments were performed where the [1] = 10[4:Zn(II)] or
10[5:Zn(II)]. Since the potentiometric titration data with the poorly
reactive, but similarly binding 6, indicate that essentially all the
phosphate is fully bound to L:Zn(II), in the absence of free catalyst
the rate of reaction should be that of the unimolecular conversion
of L:Zn:1 to product (k1cat, Scheme 1). Reactions were performed
in buffered methanol (as described above) with [L:Zn(II)] =
5.0 ¥ 10-5 mol dm-3 and [1] = 5.0 ¥ 10-4 mol dm-3. The absorbance
vs. time plots were fit to a standard first order exponential model
The constants derived from the fittings for the rate constants for
cyclization of 1 promoted by 4:Zn(II) and 5:Zn(II) as a function
s
s
s
s
cat
of pH are presented in Tables 2 and 3. The observed log k2
app
vs. pH plots for the two catalyst complexes are given in Fig. 9.
Over the sspH range studied, 4:Zn(II) shows an increase in activity
s
s
cat
up to pH ~ 10.5 at which point k2 decreases. The bell-shaped
sspH–rate data for both 4 and 5 were fit to eqn (2) (developed for
a hypothetical process with three species interconnected by two
636 | Org. Biomol. Chem., 2012, 10, 631–639
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cat
to obtain the k1 rate constants, but for very slow reactions the
(b) Cyclization of 1 catalyzed by 4:Zn(II) and 5:Zn(II)
rate constants were determined from initial rates by dividing the
slope of the linear portion of the first 5–10% of the absorbance
vs. time plots by the total expected absorbance change. The rate
constants determined from the absorbance vs. time plots were
multiplied by ten to account for the turnover number. The so-
Aside from being strongly bound to the L:Zn(II) complexes, the
cleavage of the anionic phosphate diester 1 catalyzed by 4:Zn(II)
and 5:Zn(II) is distinguished by the cooperative behaviour of two
catalyst complexes. The constants listed in Table 2 and Table 3 are
apparent second order rate constants for the reaction between the
L:Zn:1 complex and a second complex comprising one or more
cat
s
determined values of k1 as a function of pH for 4:Zn(II) and
s
5:Zn(II) are summarized in the ESI (Table 16S and Table 36S,
of the non-phosphate bound forms of the catalyst, collectively
cat
s
s
respectively†) along with plots of log k1 vs. pH (Fig. 16S and
cat
represented as [L:Zn]free (k2
in Scheme 2), as derived from
app
Fig. 36S, respectively†).
fitting the kinetic data to eqn (1). To determine if the reaction
cat
pathway involving only one molecule of catalyst (k1 in Scheme
1) contributes significantly to the overall rate, we also performed
turnover experiments using a large excess of substrate (see the ESI,
Tables 16S, 36S and Fig. 16S, 36S). Under these conditions, the
catalyst is expected to be fully bound to substrate and the observed
first order rate constant refers to conversion of the L:Zn:1 complex
(with or without added methoxide) to product (k1cat). At the sspH
maximum of 9.9 for complex 5:Zn(II), the observed rate constant
(k1cat) determined at 0.05 ¥ 10-3 mol dm-3 5:Zn(II) and 0.5 ¥ 10-3
mol dm-3 1 was 9.1 ¥ 10-4 s-1. Compared to the observed pseudo
first order rate constant of kobs = 0.15 s-1 for the cleavage of 5 ¥
10-5 mol dm-3 1 catalyzed by excess 5:Zn(II) (2.0 ¥ 10-3 mol dm-3)
at sspH = 9.9, the pathway with a single catalyst molecule is found
to account for only 0.6% of the overall rate under these conditions
A similar analysis for 4:Zn(II) operating at its optimal sspH of 12.0
shows that at [4:Zn(II)] = 1.5 ¥ 10-3 mol dm-3, only 4.7% of the
Discussion
(a) Catalyst–substrate binding
The feature of note for the kinetics of cyclization of 1 catalyzed
by 4:Zn(II) and 5:Zn(II) in methanol is the upward curvature
in the kobs vs. [catalyst] plots which signifies the involvement of
two L:Zn(II) species cooperating to promoted the cleavage of 1.
Previous reports describing the kinetics of mononuclear Zn(II)
complexes catalyzing the cleavage of phosphate diesters in water12
do not indicate there are terms greater than first order in [catalyst]
that would signify cooperation between two, non-tethered metal
centers. Comparison of the speciation diagrams of Fig. 3 and
4 clearly shows that 4 binds the Zn(II) more fully than does 5.
This is consistent with steric effects exerted by the additional
methyl groups in 5:Zn(II) that encumber the Zn(II) centre. The
binding constants of 1 to both 4:Zn(II) and 5:Zn(II), while subject
to appreciable errors from fits of the kinetic data to eqn (1), are
relatively insensitive to sspH. The speciation diagrams in Fig. 5 and
6 indicate that 4:Zn(II) and 5:Zn(II) bind 6 (and presumably 1 as
cat
reaction rate is due to the k1 pathway. For our purposes, the
unimolecular decomposition of L:Zn:1 can therefore be ignored
as a significant contributor to the reaction rate.
-
s
s
cat
its anionic ROPO2 (OAr) form) nearly completely between pH
4–9 and 6–10, respectively. Nevertheless, from the sspH/log k2
app
profiles shown in Fig. 9, the dominant reaction pathway (discussed
in more detail below) apparently involves L:Zn:1 reacting with a
second molecule of catalyst in its basic form (L:Zn(-OCH3)) up to
sspH 10–10.5, after which the catalysis drops due to the fact that
free methoxide extracts Zn2+ from the various complexes forming
Zn(-OCH3)2 or some oligomeric form thereof. With 4, the two
components of the 4:Zn(II) ꢀ 4:Zn(-OCH3) + H+ equilibrium
(sspKa = 9.76) and 4:Zn(II):6 ꢀ 4:Zn:(-OCH3):6 + H+ equilibrium
(sspKa ~10) account for ~95% of the Zn(II)-containing material
s
s
up to pH 11 and 12, respectively, above which formation of
Zn(-OCH3)2 becomes prominent. With 5, which binds Zn(II) some-
what weaker than does 4, the 5:Zn(II) ꢀ 5:Zn:(-OCH3) + H+ ꢀ
Zn(-OCH3)2 equilibrium is such that nearly equal concentrations
The cyclization of 1, bound as a L:Zn:1 complex that reacts with
a second 4:Zn(II) or 5:Zn(II), is significantly accelerated relative to
the methoxide-promoted background reaction of 1. The maximal
second order rate constants exhibited by 4:Zn(II) and 5:Zn(II),
as determined from the fits in Fig. 9 (23.1 2.5 dm3 mol-1 s-1
and 146.1 30.0 dm3 mol-1 s-1, respectively), are over 104 greater
s
of these three components exist at pH 10.2, with [Zn(-OCH3)2]
s
increasing sharply at higher values. Interestingly, the presence
of phosphate tends to stabilize the 5:Zn:6 complexes so that
the two components of the 5:Zn(II):6 ꢀ 5:Zn:(-OCH3):6 + H+
equilibrium (sspKa ~11) comprise about 85–90% of the phosphate-
s
s
containing species up to pH 12. The complexity of speciation
than the second order rate constant for the methoxide-promoted
s
s
OMe
at high pH indicates that it is an oversimplification to describe
cleavage of 1 (k2
= 2.56 ¥ 10-3 dm3 mol-1 s-1).22 The addition of
the kinetic behaviors shown in Fig. 9 in terms of a process
the methyl substituents in 5:Zn(II) provides at most a factor of five
rate enhancement compared to 4:Zn(II). This can be compared
with the results of a similar study of the cleavage of 1 promoted by
dinuclear complexes15 where the dinuclear complex 8:Zn(II)2 was
over 10 times more reactive than complex 7:Zn(II)2 and complex
10:Zn(II)2 was over 103 more reactive than 9:Zn(II)2.
s
involving just three species interconnected by two pKa values,
s
particularly at high sspH where Zn(-OCH3)2 becomes an important
species. Nevertheless, this minimalistic approach serves to describe
s
s
the kinetic behaviour on the low pH side up to the plateau
region.
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plots observed for 7:Zn(II)2 and 8:Zn(II)2, and were previously
determined to be 1.6 ¥ 103 dm3 mol-1 s-1 and 29.8 ¥ 103 dm3 mol-1
s-1, respectively.15 The EM values based on this analysis are thus
EM4 = 7 ¥ 10-3 mol dm-3 and EM5 = 2 ¥ 10-1 mol dm-3.
The low values of EM obtained here for 4:Zn(II) and 5:Zn(II)
can be interpreted in one of two ways: (1) a poor dinuclear
catalyst (small k2, Scheme 2); or (2) more likely, a very effective
cooperativity in the mononuclear catalyst (large k3, Scheme 2).
s
s
To address the first possibility, under optimal pH conditions
(sspH = 8.0), the saturating rate constant for the unimolecular
decomposition of 8:Zn(II)2:1 was previously15 found to be kcat
=
max
6.2 s-1 while in the present study, the unimolecular decomposition
s
s
cat
of the 5:Zn(II):1 complex at the optimum pH of 10.8 is k1
=
0.0023 s-1 (ESI, Table 36S). This comparison shows that when
bound to substrate 1, 5:Zn(II) is nearly 2700-fold less active than
the dinuclear species.
The low values of EM for 4:Zn(II) and 5:Zn(II) must therefore
result from the high efficiency of the cooperative behaviour of
the two mononuclear complexes. The binding of the first L:Zn(II)
catalyst to the substrate, while not producing a very active form
of L:Zn:1, activates the bound substrate in the latter toward
further catalysis by L:Zn(II). This is particularly evident in the
case of 4:Zn(II) where the computed EM (7 ¥ 10-3 mol dm-3)
lies well within a practically attainable concentration range. This
suggests that, unlike the situation in water, where no catalyst
cooperativity has ever been observed, the use of methanol as
a solvent seems to predispose mononuclear Zn(II) complexes to
cooperate in the cleavage of diester 1, generating a catalytic system
which approaches the efficiency of dinuclear systems where the two
metal ions are physically connected.
Unfortunately, it is difficult to compare our EM values with
those for other synthetic catalysts in water since no reports are
available of mononuclear systems that exhibit such cooperativity.
In comparison to our own previous work, the EM values for the
catalytic systems reported here are much lower than the one that we
now compute for complex 2:Zn(II), the first mononuclear catalyst
for which we observed evidence for catalyst cooperativity.6a
Comparing the second order rate constant for the cleavage of
1 catalyzed by 3:Zn(II)2 (2.75 ¥ 105 dm3 mol-1 s-1) with the third
order rate constant for the reaction of two equivalents of 2:Zn(II)
with substrate 1 (1.8 ¥ 103 dm6 mol-2 s-1), gives EM = 153 mol
dm-3. The very large EM for 2:Zn(II) is a function of the very large
catalysis afforded by the dinuclear catalyst and the much weaker
cooperativity of the mononuclear complex. Unlike the situation
with 4:Zn(II) and 5:Zn(II), the efficiency of the cooperativity with
2:Zn(II) is limited by poor binding to substrate 1, since saturation
binding between this substrate and 2:Zn(II) is not observed.
Scheme 2 Comparative systems for determining effective molarity (EM).
(c) Effective molarities of 4:Zn(II) and 5:Zn(II)
A comparison of the catalysis afforded by complexes 4:Zn(II)
and 5:Zn(II) with that provided by the respective dinuclear
counterparts (7:Zn(II)2 and 8:Zn(II)2) quantifies the cooperative
mechanism in terms of the effective molarity (EM). As generally
applied, EM describes the efficiency of a system exhibiting an
intramolecular unimolecular catalytic effect relative to a bimolec-
ular process and is defined as the quotient of the first order rate
constant for the intramolecular reaction divided by the second
order rate constant for the intermolecular reaction proceeding
by a comparable mechanism.23 EM has units of concentration
and represents the hypothetical [catalytic species] required for
the intermolecular reaction to occur at the same rate as the
intramolecular one. EM values as high as 1010 mol dm-3 23c
are obtained for intramolecular nucleophilic catalysis, while for
intramolecular general base catalysis the EM is generally much
lower with limiting values of 100 mol dm-3, but most examples
being < 1.0 mol dm-3.24
The EM values here are computed in a slightly different
way, comparing the apparent second order rate constants for
the reaction of the dinuclear catalysts (7:Zn(II)2 and 8:Zn(II)2)
with substrate 1 (k2, Scheme 2) with the apparent third order
rate constant for the reaction between 1 and two molecules of
the mononuclear catalysts (4:Zn(II) and 5:Zn(II)) (k3 in Scheme
2). These EM values still represent the concentration of the
mononuclear catalyst that would be necessary to achieve the same
reaction rate as the analogous dinuclear complex. The third order
rate constants for the reaction between 4:Zn(II) and 5:Zn(II) with
cat
cat
max
L:Zn:1 are defined as k3 = (k2
)
max/Kd where (k2
)
is the
app
app
Conclusion
maximal second order rate constant for the reaction between the
L:Zn:1 complex and a second molecule of L:Zn(II) obtained by
fitting the data in Tables 1 and 2 to eqn (2). Kd refers to the average
catalyst:substrate dissociation constant (averaged from data in
Tables 2 and 3). The respective third order rate constants (k3) for
4:Zn(II) and 5:Zn(II) with L:Zn:1 are (23.1 dm3 mol-1s-1)/(1.0 ¥ 10-4
mol dm-3) = 2.3 ¥ 105 dm6 mol-2 s-1 and (146.1 dm3 mol-1s-1)/(1.0 ¥
10-3 mol dm-3) = 1.5 ¥ 105 dm6 mol-2 s-1, respectively, at the
The present study in methanol, and previous ones from our labs
concerning the metal ion promoted cleavage of phosphate diesters
in alcohol, might be criticized for the apparent failing of the solvent
model of an alcohol relative to the apparent situation in enzymes
where the gross medium is water. This criticism can be challenged
by pointing out that numerous biological processes involving acyl
or phosphoryl transfer processes are really transesterifications,
as exemplified by cleavages of RNA where a 2¢-hydroxyl on a
ribose is deprotonated to attack a 3¢-phosphate cleaving it to a
2¢,3¢-cyclic phosphate. Secondly, the active site of the enzymes
s
s
maxima of their respective pH/rate profiles. The second order
rate constants for the reaction of the dinuclear catalysts with 1
are defined as k2 = kcat/KM, derived from the Michaelis–Menten
638 | Org. Biomol. Chem., 2012, 10, 631–639
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promoting such reactions have reduced polarities, akin to those of
the light alcohols, as well as being quite deficient in water content,
thus being more like a molecular bottle, the insides of which are
adorned with specific functional groups that are important to the
catalytic process at hand. In the simplified biomimetic examples,
the intramolecular cyclization of 1 serves as a highly simplified
model for numerous studies in water and also for our studies in
alcohol, where the major effects are greater binding constants
promoted by the medium, and also very much lower transition
state energies for the metal catalyzed phosphoryl transfer reaction.
We previously rationalized the large rate accelerations observed
for the cleavage of phosphate diesters catalyzed by dinuclear
Zn(II) complexes in methanol6,14,15 on the basis of several factors,
the most important of which are: (1) enhanced association of
the metal complex and substrate; (2) double activation of the
substrate through Zn(II) ◊ ◊ ◊ O-–P(OR)OAr)–O ◊ ◊ ◊ Zn(II) binding;
and (3) lowering the activation barrier for the transesterification
by a synergistic medium effect that stabilizes a transition state of a
transforming Zn(II)2-bound substrate where there is more charge
dispersal than in the ground state. The current study highlights
additional, less appreciated but none-the-less beneficial aspects of
alcohol solvent, namely promoting cooperative behaviour among
mononuclear Zn(II) complexes. The observation of strong binding
between a mononuclear Zn(II) complex and a phosphate diester
is rare in water, but apparently far more common in a reduced
dielectric alcohol medium such as methanol or ethanol.25 We
propose here an additional and perhaps far more common than
realized aspect, namely the emergence of cooperative mechanisms
involving initial strong binding between the metal complexes and
anionic substrates like 1 followed by transient association of a
second molecule of catalyst to induce the catalytic transformation
of the substrate.
T. Liu, S. E. Bunn, D. Edwards, C. J. White, S. A. Melnychuk and R.
S. Brown, J. Am. Chem. Soc., 2008, 130, 6639.
6 R. S. Brown and A. A. Neverov, J. Chem. Soc., Perkin Trans. 2, 2002,
1039.
7 J. S. Tsang, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc., 2003,
125, 1559.
8 For a representative list of references on various dinuclear metal-
containing complexes, see: (a) J. Weston, Chem. Rev., 2005, 105, 2151;
(b) N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res.,
1999, 32, 485; (c) G. Feng, D. Natale, R. Prabaharan, J. C. Marque-
Rivas and N. H. Williams, Angew. Chem., Int. Ed., 2006, 45, 7056;
(d) M.-Y. Yang, O. Iranzo, J. P. Richard and J. R. Morrow, J. Am.
Chem. Soc., 2005, 127, 1064; (e) O. Iranzo, T. Elmer, J. P. Richard
and J. R. Morrow, Inorg. Chem., 2003, 42, 7737; (f) O. Iranzo, J.
P. Richard and J. R. Morrow, Inorg. Chem., 2004, 43, 1743; (g) O.
Iranzo, A. Y. Kovalevsky, J. R. Morrow and J. P. Richard, J. Am. Chem.
Soc., 2003, 125, 1988; (h) F. Mancin, E. Rampazzo, P. Tecilla and U.
Tonellato, Eur. J. Org. Chem., 2004, 281; (i) M. Subat, K. Woinaroschy,
C. Gerstl, B. Sarkar, W. Kaim and B. Ko¨nig, Inorg. Chem., 2008, 47,
4661; (j) H. Linjalahti, G. Feng, J. C. Marque-Rivas, S. Mikkola and
N. H. Williams, J. Am. Chem. Soc., 2008, 130, 4232.
9 (a) R. S. Brown and A. A. Neverov, Adv. Phys. Org. Chem., Ed: J. P.
Richard, Elsevier: San Diego, Calif, 2007, 42, 271; (b) R. S. Brown, Z.-l.
Lu, C. T. Liu, W. Y. Tsang, D. R. Edwards and A. A. Neverov, J. Phys.
Org. Chem., 2009, 23, 1; R. S. Brown, Progress in Inorganic Chemistry,
Ed: K. D. Karlin, John Wiley & Sons, 2012, 57, 55.
10 J. Kim and H. Lim, Bull. Korean Chem. Soc., 1999, 20, 491.
11 (a) T. Gadja, T. Du¨pre, I. To¨ro¨k, J. Harmer, A. Schweiger, J. Sander, D.
Kuppert and K. Hegetschweiler, Inorg. Chem., 2001, 40, 4918; (b) A.
Jancso´, S. Mikkola, H. Lo¨nnberg, K. Hegetschweiler and T. Gadja,
Chem.–Eur. J., 2003, 9, 5404.
12 For a representative list of references on various mononuclear metal
containing complexes, see: (a) M. Livieri, F. Mancin, U. Tonellato and
J. Chin, Chem. Commun., 2004, 2862; (b) G. Feng, J. C. Marque-Rivas
and N. H. Williams, Chem. Commun., 2006, 1845; (c) M. Livieri, F.
Mancin, G. Saielli, J. Chin and U. Tonellato, Chem.–Eur. J., 2007, 13,
2246; (d) R. Bonomi, G. Saielli, U. Tonellato, P. Scrimin and F. Mancin,
J. Am. Chem. Soc., 2009, 131, 11278; (e) L. Bonfa, M. Gatos, F. Mancin,
P. Tecillaand and U. Tonellato, Inorg. Chem., 2003, 42, 3943; (f) T. Koike
and E. Kimura, J. Am. Chem. Soc., 1991, 113, 8935.
13 K. A. Deal and J. N. Burstyn, Inorg. Chem., 1996, 35, 2792.
14 M. F. Mohamed, A. A. Neverov and R. S. Brown, Inorg. Chem., 2009,
48, 11425.
15 M. F. Mohamed and R. S. Brown, J. Org. Chem., 2010, 75, 8471.
16 W. Y. Tsang, D. R. Edwards, S. A. Melnychuk, C. T. Liu, C. Liu, A.
A. Neverov, N. H. Williams and R. S. Brown, J. Am. Chem. Soc., 2009,
131, 4158.
Acknowledgements
The authors gratefully acknowledge the generous support of the
Natural Sciences and Engineering Research Council of Canada
(NSERC). In addition M. F. M. acknowledges the receipt of an
NSERC Alexander Graham Bell postgraduate scholarship (CGS-
D). The authors are grateful to Mr Scott Strum who performed
some preliminary experiments.
17 P. C. Kunz, N. E. Bru¨ckmann and B. Spingler, Eur. J. Inorg. Chem.,
2007, 394.
18 J. Zhang, G. Leitus, Y. Ben-Davidand and D. Milstein, J. Am. Chem.
Soc., 2005, 127, 10840.
19 B. de Bruin, J. A. Brands, J. J. J. M. Donners, M. P. J. Donners, R. de
Gelder, J. M. M. Smits, A. W. Gal and A. L. Spek, Chem.–Eur. J., 1999,
5, 2921.
References
20 (a) G. Gibson, A. A. Neverov and R. S. Brown, Can. J. Chem., 2003, 81,
495; (b) For the designation of pH in non-aqueous solvents we use the
nomenclature recommended by the IUPAC, Compendium of Analytical
Nomenclature. Definitive Rules 1997 3rd edn, Blackwell, Oxford, UK,
1998. The pH meter reading for an aqueous solution determined with
an electrode calibrated with aqueous buffers is designated as wwpH; if
the electrode is calibrated in water and the ‘pH’ of the neat buffered
methanol solution then measured, the term swpH is used; and if the
electrode is calibrated in the same solvent and the ‘pH’ reading is made,
1 G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams and R. Wolfenden,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 4052.
2 (a) W. N. Lipscomb and N. Stra¨ter, Chem. Rev., 1996, 96, 2375; (b) J.
A. Cowan, Chem. Rev., 1998, 98, 1067; (c) W. W. Cleland and A. C.
Hengge, Chem. Rev., 2006, 106, 3252; (d) N. Stra¨ter, W. N. Lipscomb, T.
Klabunde and B. Krebs, Angew. Chem., Int. Ed. Engl., 1996, 35, 2024;
(e) D. E. Wilcox, Chem. Rev., 1996, 96, 2435; (f) R. Kra¨mer, Coord.
Chem. Rev., 1999, 182, 243.
3 For some recent reviews see: (a) F. Mancin and P. Tecilla, New J. Chem.,
2007, 31, 800; (b) J. R. Morrow, Comments Inorg. Chem., 2008, 29, 169;
(c) M. Livieri, F. Mancin, G. Saielli, J. Chin and U. Tonellato, Chem.–
Eur. J., 2007, 13, 2246.
4 (a) W. W. Cleland, P. A. Frey and J. A. Gerlt, J. Biol. Chem., 1998,
273, 25529; (b) T. Simonson, F. Carlsson and D. A. Case, J. Am. Chem.
Soc., 2004, 126, 4167 and references therein; (c) J. P. Richard and T. L.
Amyes, Bioorg. Chem., 2004, 32, 354.
5 (a) A. A. Neverov, Z.-L. Lu, C. I. Maxwell, M. F. Mohamed, C. J. White,
J. S. W. Tsang and R. S. Brown, J. Am. Chem. Soc., 2006, 128, 16398;
(b) S. E. Bunn, C. T. Liu, Z.-L. Lu, A. A. Neverov and R. S. Brown, J.
Am. Chem. Soc., 2007, 129, 16238; (c) C. T. Liu, A. A. Neverov and R.
S. Brown, J. Am. Chem. Soc., 2008, 130, 16711; (d) A. A. Neverov, C.
s
s
then the term pH is used. In methanol swpH-(-2.24) = pH and since
s
s
the autoprotolysis constant of methanol is 10-16.77, neutral sspH is 8.4.
21 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739.
22 J. S. Tsang, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc., 2003,
125, 1559.
23 (a) A. J. Kirby and F. Hollfelder, From Enzyme Models to Model
Enzymes, RSC Publishing, Cambridge, 2009, 1st edn, pp. 17–18; (b) M.
I. Page, Chem. Soc. Rev., 1973, 2, 295; (c) M. I. Page and W. P. Jencks,
Proc. Natl. Acad. Sci. U. S. A., 1971, 68, 1678.
24 A. J. Kirby and G. J. Lloyd, J. Chem. Soc., Perkin Trans. 2, 1976,
1753.
25 C. Tony Liu, Alexei A. Neverov and R. Stan Brown, Inorg. Chem., 2007,
46, 1778.
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