Enzyme-like acceleration for the hydrolysis of a DNA model promoted by a dinuclear Zn(II) catalyst in dilute aqueous ethanol

Article abstract of DOI:10.1021/ja805801j

The rates and products of cleavage of methyl (2-chloro-4-nitrophenyl) phosphate (2) promoted by a dinuclear Zn(II) complex (3) of 1,3-bis-N,N′(1,5,9-triazacyclododecyl)propane along with 1 equiv of ethoxide were investigated in ethanol solution containing small amounts of water (8 mM ≤ [H₂O] ≤ 2.1 M) at 25 °C and ^s_spH 7.9. The kinetics of decomposition of 5 × 10^-5 M 2 in the presence of varying [3] (0.03 mM ≤ [3] ≤ 0.12 mM) in ethanol containing 28 mM water show very strong saturation binding which is analyzed to give fitted values for the complex dissociation constant (K_d) and maximum catalytic rate constant (k_cat^max) of 3.2 × 10^-8 M and 1.47 × 10^-3 s^-1. Product analysis indicates that the reaction in ethanol containing 28 mM and 2.1 M water produces 46% and 93% of the hydrolysis product (methyl phosphoric acid), with the remainder being ethanolysis product (ethyl methyl phosphate). Analysis of the kinetics of the reaction at 28 mM water indicates that second order rate constant for the catalytic reaction, given as k_cat^max/K_d, is 4.6 × 10⁴ M^-1 s^-1, is a factor of 8.4 × 10¹⁰ larger than the second order rate constant (k₂^EtO) for the ethoxide promoted reaction of 2 in ethanol (5.5 ± 0.3) × 10^-7 M^-1 s^-1. A more detailed analysis that considers the relative acidities of water and ethanol in ethanol solvent indicates that at ^s_spH 7.9, the ethanolysis is accelerated by 2.3 × 10¹⁴ times relative to the computed ethoxide reaction at that ^s_spH, while the hydrolysis isaccelerated by ≥1.6 × 10¹⁷ times relative to the background hydroxide reaction, suggesting that complex 3 promotes the hydrolysis at least 1000 times more effectively than ethanolysis. Copyright

Full text of DOI:10.1021/ja805801j

Published on Web 09/27/2008
Enzyme-like Acceleration for the Hydrolysis of a DNA Model Promoted by a
Dinuclear Zn(II) Catalyst in Dilute Aqueous Ethanol
C. Tony Liu, Alexei A. Neverov, and R. Stan Brown*
Department of Chemistry, Queen’s UniVersity, Kingston, Ontario, Canada, K7L 3N6
Received July 24, 2008; E-mail: rsbrown@chem.queensu.ca
Molecules containing phosphate diester moieties (RO)PO2-(OR′)
serve important biological functions because of their inherent
stability toward hydrolytic and nucleophilic cleavage, making this
functional group ideal for the preservation of genetic information
in RNA and DNA.1 Several enzymes that cleave RNA and DNA
contain metal ions such as Zn2+,2 and considerable research has
been directed at understanding the mechanisms of hydrolysis and
alcoholysis of RNA, DNA, and their model systems,2,3 as well as
toward designing M2+-containing catalysts for facilitating their
cleavage.4
We recently showed that the cleavages of the RNA and DNA
models 1 and 2 promoted by the Zn(II)2 complex 35,6 in methanol
and ethanol are accelerated by 1012 times or more relative to
Figure 1. A plot of the observed first order rate constant for cleavage of
2a (5 × 10-5 M) in ethanol with 28 mM H2O vs varying [3], T ) 25 °C,
the background methanolysis reactions. In water, complex 3b
sspH ) 7.90.10,11 The small apparent x-intercept of ∼1.7 × 10-5 M is
attributed to a dissociation of Zn2+ from 3 at very low concentrations.5,6,9
is no more active than the mono-Zn(II) complex of 1,5,9-
triazacyclododecane toward the cleavage of phosphate diesters,7
and we surmised that the acceleration of catalysis in methanol
is attributable to a medium effect of reduced dielectric constant
and polarity.5,6 The catalyzed cleavages in methanol, although
fast, are transesterifications and not hydrolytic reactions. In the
case of the RNA models, the first formed product arises from
intramolecular ring closure as it does in water, but in the DNA
model cases the OAr leaving group is replaced by OR (Scheme
1).
M) of water in ethanol leads to 93:7 ratio of the hydrolysis
product (4) to ethanolysis product (5).
(CH3O)PO32- + (CH3O)(CH3CH2O)PO-2
4
5
There are two main points of note. First, the plot in Figure 1 of
the observed rate constant for cleavage of 2a (5 × 10-5 M added
as the acid) promoted by varying [3] with added equimolar NaOEt
in anhydrous ethanol (used as supplied, but contains 28 mM H2O
by Karl Fischer titration) shows very strong 3 + 2a saturation
binding, followed by a chemical step liberating the phenol.9
Nonlinear least-squares fitting of the data to a universal binding
equation5,6,9 gives the computed line through the data. The fit is
based on an upper limit for the complex dissociation constant (Kd)
of 3.2 × 10-8 M (determined iteratively by varying the Kd until
the goodness of fit did not change9) and maximum catalytic rate
constant for release of 2-Cl-4-NO2-phenol (kcatmax) of 1.47 × 10-3
s-1. The apparent second order rate constant for the catalyzed
reaction (k2cat), given as kcatmax/Kd, is g4.6 × 104 M-1 s-1 which
is a factor of at least 8.4 × 1010 larger than the second order rate
constant (k2EtO) for the ethoxide promoted reaction of 2a in ethanol
Scheme 1
The X-ray structure of 3b with a bridging HO- was
determined5c as grown from methanol solution at ambient
conditions. The X-ray structure of the di-Cu(II) complex of the
same ligand, also grown at ambient temperature, shows both a
bridging hydroxide and a bridging water, the latter being replaced
by a bridging phosphate when the crystal is grown in the
presence of dibenzyl phosphate.5d This suggests that the catalyst
might have a special affinity for a bridging HO- ion, so under
appropriate conditions the medium effect might be harnessed to
accelerate the 3-promoted hydrolytic reactions of phosphate
diesters.8 We report here a realization of this goal obtained
through a study of the cleavage of the DNA model 2a promoted
by 3 in ethanol which reveals not only a large rate acceleration
but an interesting phenomenon where as little as 3.8 vol % (2.1
(5.5 ( 0.3) × 10-7 M-1 s-1 9
.
Second, the products of cleavage of 2a mediated by 3 in ethanol
containing varying, but small, amounts of water (8 mM e [H2O]
1
e 2.1 M) were quantitatively determined by H NMR analysis of
the ethoxy, methoxy, and phenol peaks of the products isolated
after reaction and confirmed by MS and 31P NMR (see Table S2;
sample NMR spectra are presented in Figures S1-S49). In Figure
2 is a plot of the % hydrolytic product (4) as a function of added
H2O. To complement the above product determinations, the kinetics
of the cleavage of 2.5 mM 2a promoted by 2.5 mM 3 in ethanol
containing 1 equiv of NaOEt were investigated at a few [H2O].
The results, shown in Figure 3 (data tabulated in Table S7,
Supporting Information), indicate a drop of slightly more than a
9
13870 J. AM. CHEM. SOC. 2008, 130, 13870–13872
10.1021/ja805801j CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Charges Omitted for Clarity
so we must rule out reasonable alternatives such as attack of
hydroxide on the 3:2a complex. On first inspection, one might
expect, on the basis of autoprotolysis constants, that water (KW
)
10-14) is more acidic than ethanol (KE ) 10-19.1).11 In mixed
solvents, this ignores the medium effect of transferring the
dissociating water into a less polar medium so it is expected to be
less dissociated in ethanol than in water. However, the exact
calculation of the effects requires knowledge of how the various
equilibria and activity coefficients depend on the mixed solvent
which is not known at the present time.13
Figure 2. A plot of the percentage of analyzed hydrolysis product
(CH3OPO32-) produced from the reaction of 2.5 mM 2a promoted by 2.5
mM 3 in ethanol with varying amounts of water at room temperature.
Fortunately, we can experimentally estimate the ionization
constant of water in ethanol. In a little known, but important paper,
Caldin and Long14 presented evidence that in ethanol, or ethanol
containing “not more than a few percent water”, the acid dissocia-
tion constants of ethanol and water are similar with water being
slightly weaker. We confirmed this by determining the rate constants
and cleavage products of cleavage of p-nitrophenyl benzoate under
basic conditions in ethanol containing 0.53 and 1.03 M water where
the amounts of hydrolysis products are 1.0 and 2.3% (Tables
S2-S5, Supporting Information9). Using a computational approach
based on that provided,14 we find that the ratio between the acid
dissociation constant of water and ethanol (ssKaw/ssKaE)9 in ethanol
Figure 3. A plot of the observed first order rate constant for cleavage of
2a (2.5 mM) in ethanol with 2.5 mM of 3 in ethanol vs varying [H2O] in
ethanol at T ) 25 °C. The rate constants were determined from initial rate
methods where the first 5-10% of the abs vs time trace for the appearance
of the phenolic product at 323 nm was fitted to a standard linear regression
model.
at these two water concentrations is 0.83 and 0.91. Assuming a
s
linear correlation of the ratio with [water], Kaw/ssKaE ) 0.75 was
s
computed for ethanol containing 28 mM water, showing water to
be slightly less acidic than ethanol at the concentrations employed,
in agreement with what Caldin and Long determined.14 The acid
dissociation constant of ethanol containing 28 mM H2O is assumed
factor of 2 in passing from 16 mM to 1 M water, probably due to
a change in the medium since it was previously determined that
complex 3 has an unremarkable reactivity in an aqueous solution.7
One also sees that the rate constant for the catalyzed reaction at
the 25-times higher concentration of catalyst is about three times
lower than under the conditions of Figure 1, probably due to the
inhibitory effect of triflate counterions.5,6
to be reliably computed from the autoprotolysis constant of pure
s
ethanol as pKaE ) -log(10-19.1/[EtOH]) ) 20.33 while that of
s
water (sspKaw) in the same medium is 20.45.
The so-determined acidities of water and ethanol indicate that
free -OH cannot be the active nucleophile in hydrolyzing 3:2a in
ethanol under the kinetic conditions of Figure 1. The exact treatment
is shown in section 2a of the Supporting Information, but is
The unusual points brought to light in this study are the large
amount of hydrolysis and increase in rate of the hydrolytic rates
brought about by the combination of a dinuclear Zn(II) catalyst
and a medium effect in ethanol. That as little as 28 mM of water
(0.05 vol%), in the presence of an overwhelming excess of ethanol
gives 46% of hydrolysis product from a relatively inert phosphodi-
ester suggests a process that selects for H2O or -OH (either external
or di-Zn(II) complex-coordinated) relative to ethanol or ethoxide
as the active nucleophile attacking the 3:2a complex. Solvolytic
reactions in mixed ethanol/water media are known to be complex12
but the available evidence allows us to rule out external H2O and
-OH as being responsible for the hydrolysis, leaving complex 3b
with an intramolecularly coordinated HO- as the most likely
catalyst.
summarized as follows. At sspH ) 7.9 and [H2O] ) 0.028 M, [OH-]
s
) Kaw ([H2O]/[H+]) ) 7.9 × 10-15 M. To account for the fact
s
that 46% of the reaction at this water content gives hydrolysis
product, the rate constant for external -OH attack would be (0.46
× kcatmax)/[HO-] ) 8.6 × 1010 M-1 s-1 which exceeds the
diffusion limit in ethanol by a factor of 8.6.15 By exclusion, the
active species is most simply formulated as 3b, or some closely
related complex having an asymmetrical doubly coordinated or
singly coordinated HO-.
Given in Scheme 2 is a proposed pathway which is common to
3b and 3c catalyzed cleavage of 2a. The scheme builds on our
previous interpretation that the substrate becomes doubly activated
through binding to both Zn(II) ions followed by an intramolecular
delivery of the coordinated nucleophile (ethoxide or hydroxide).
Structure 6 in the scheme is proposed by analogy to the X-ray
structure of the di Cu(II) complex where a bridging dibenzyl
phosphate and a bridging hydroxide are observed to complete the
five-coordinate metal ion ligation.5d Whether the actual attack is
stepwise, through a phosphorane intermediate, or concerted cannot
be established with the information at hand.3,6a
Our earlier study of the 3-catalyzed methanolysis5b of methyl
p-nitrophenyl phosphate indicated that the rate maximized at a 1:2:1
ratio of ligand/Zn2+/-OCH3, suggesting that the optimally effective
catalyst is 3a. The same is seen for the 3-catalyzed reaction of 2a
in ethanol9,6b indicating that the transition state contains a 1:2:1:1
ratio of ligand, Zn(II), phosphate, and lyoxide (EtO- and/or HO-
or its chemical equivalent). This assertion requires that the species
leading to the hydrolysis product is a complex-coordinated HO-,
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C O M M U N I C A T I O N S
It has been stated that “the active sites of enzymes are
non-aqueous, and the effective dielectric constants resemble those
in organic solvents rather than that in water”.16 The low dielectric
interior of enzymes also means that ion-dipole and ion-ion
interactions will be stronger than in water17 and thus might provide
a very effective way to lower the transition state energies for metal
promoted reactions of anionic substrates. However, it is difficult
to quantify the effect given the complexity of the enzyme catalyzed
processes. The present data indicate that the reduced dielectric
constant of ethanol relative to water (24.3 vs 78) plays an important
role in achieving the acceleration for the hydrolytic process observed
here with a rather simple dinuclear Zn(II) complex. Just how great
is the acceleration can be quantified in two simple ways,9 comparing
the second order rate constants (kcatmax/Kd) for the catalytic reaction
and those for the lyoxide reactions or by comparing the kcatmax value
3 shows a very large selectivity for activating water as a nucleophile
in the presence of an overwhelming concentration of ethanol. These
results demonstrate in a convincing way an underappreciated mode
by which very large rate accelerations for hydrolytic reactions might
be achieved by the coupling of catalytically important functional
groups and medium effects.
Acknowledgment. The authors acknowledge financial assistance
of NSERC, the Canada Council for the Arts through the award of
a Killam Research fellowship to RSB, and Queen’s University. In
addition they thank Prof. E. Bosch, Prof. M Rose´s, and Prof. J. P
Guthrie for helpful discussions and the referees of the initial
manuscript for their constructive criticisms. C.T.L. thanks NSERC
for a PGSD Scholarship.
Supporting Information Available: Experimental details, support-
ing text, Figures S1-S6, Tables S1-S7. This material is available free
of charge via the Internet at http://pubs.acs.org.
for cleavage of the 3:2a complex to the background lyoxide
s
s
reactions at pH ) 7.90. For the first method, the 54:46 ratio of
the products 5:4 at 28 mM H2O, requires k2cat (ethanolysis) ) 0.54
× (kcatmax/Kd) ) 2.48 × 104 M-1 s-1 and k2cat (hydrolysis) ) 2.11
× 104 M-1 s-1. Initial rate experiments indicate that the rate
constant for the lyoxide reaction does not increase with increasing
[H2O], so that an upper limit for the k2 value for the hydroxide
reaction is approximately that of the ethoxide reaction in the absence
of catalyst (see Supporting Information). Thus, the accelerations
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