Catalytic zinc complexes for phosphate diester hydrolysis

Article abstract of DOI:10.1002/anie.201400335

Creating efficient artificial catalysts that can compete with biocatalysis has been an enduring challenge which has yet to be met. Reported herein is the synthesis and characterization of a series of zinc complexes designed to catalyze the hydrolysis of phosphate diesters. By introducing a hydrated aldehyde into the ligand we achieve turnover for DNA-like substrates which, combined with ligand methylation, increases reactivity by two orders of magnitude. In contrast to current orthodoxy and mechanistic explanations, we propose a mechanism where the nucleophile is not coordinated to the metal ion, but involves a tautomer with a more effective Lewis acid and more reactive nucleophile. This data suggests a new strategy for creating more efficient metal ion based catalysts, and highlights a possible mode of action for metalloenzymes.

Full text of DOI:10.1002/anie.201400335

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Angewandte
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DOI: 10.1002/anie.201400335
Enzyme Models
Catalytic Zinc Complexes for Phosphate Diester Hydrolysis**
Emmanuel Y. Tirel, Zoꢀ Bellamy, Harry Adams, Vincent Lebrun, Fernanda Duarte, and
Nicholas H. Williams*
Abstract: Creating efficient artificial catalysts that can compete
with biocatalysis has been an enduring challenge which has yet
to be met. Reported herein is the synthesis and characterization
of a series of zinc complexes designed to catalyze the
hydrolysis of phosphate diesters. By introducing a hydrated
aldehyde into the ligand we achieve turnover for DNA-like
substrates which, combined with ligand methylation, increases
reactivity by two orders of magnitude. In contrast to current
orthodoxy and mechanistic explanations, we propose a mech-
anism where the nucleophile is not coordinated to the metal
ion, but involves a tautomer with a more effective Lewis acid
and more reactive nucleophile. This data suggests a new
strategy for creating more efficient metal ion based catalysts,
and highlights a possible mode of action for metalloenzymes.
coordinated nucleophiles to enhance the attack at phospho-
rus. Chin and co-workers established that the effectiveness of
this nucleophile can depend strongly on ligand structure.[4] If
this nucleophile is part of the ligand structure, then its
efficiency can be enhanced through careful design, and
substantial rate enhancements achieved compared to that
a metal-bound hydroxide. However, the flaw in this strategy is
that the product is a phosphorylated ligand which is very
stable, and so the complexes are not catalytic.
A potential solution to this problem is suggested by the
hydrolysis of model compounds also containing keto or
aldehyde groups.[5] Bender and Silver showed that benzoate
ester hydrolysis can be accelerated 105-fold by the presence of
an ortho aldehyde group. This hydrate form of the aldehyde
provides an effective nucleophile, thus producing a product
which can readily decompose to reform the carbonyl.[6]
Similar effects have been reported for phosphate ester
cleavage.[7] To create a catalytic system, Menger and Whitesell
incorporated aldehydes into micellar head groups, and these
aggregates showed both enhanced activity and turnover.[8]
Interestingly, recent work with sulfatases and phosphonohy-
drolases has shown that a formyl glycine residue in the active
site is believed to act as a nucleophile through its hydrated
form. It has been speculated that this nucleophile may
facilitate the broad substrate tolerance of these enzymes as
the covalently modified enzyme can decompose through
a common mechanism (reforming the aldehyde by eliminat-
ing the derivatized hydroxy) which is independent of the
functional group being hydrolyzed.[9]
S
ubstantial efforts have been made to create metal ion
complexes that are effective catalysts for phosphate ester
hydrolysis.[1] These compounds provide insight into how
biological catalysts might function, and hold the promise of
creating novel therapeutics or laboratory agents for manip-
ulating nucleic acids.[2] The challenges of sufficient activity to
function usefully under biological conditions and achieving
turnover remain. Herein we report how incorporating
a hydrated aldehyde as a nucleophile can enhance reactivity
and lead to turnover. Our mechanistic explanation provides
a new strategy for designing metal ion complexes with
nuclease activity.
In developing artificial metal ion complexes to cleave
RNA, the 2’OH group provides an intramolecular nucleo-
phile which can be exploited.[3] For DNA, this is not possible,
and the most effective strategies to date have used metal-ion-
Our designs are based on pyridyl zinc complexes with
a simple alcohol chain as a nucleophile (1; Scheme 1). The
propylene linker is much more reactive than the ethylene
analogue, or complexes which do not have an alkoxy
nucleophile. It has been shown that 2-amino substituents on
the pyridyl ring can have a large effect on reactivity, and is
presumed to be due to potential hydrogen bonding with the
substrate.[10] We decided not to incorporate an amino group in
this work so as to avoid condensation reactions with the
aldehyde. Instead, we incorporated methyl groups into the 2-
[*] E. Y. Tirel, Z. Bellamy, H. Adams, V. Lebrun, Prof. N. H. Williams
Department of Chemistry
Sheffield University, Sheffield (UK)
E-mail: N.H.Williams@Sheffield.ac.uk
F. Duarte
Department of Cell and Molecular Biology
Uppsala University, Uppsala (Sweden)
[**] Financial support from the Engineering and Physical Sciences
Research Council (EP/E01917X) and European Commission (ITN
PhosChemRec 238579) is gratefully acknowledged. We would also
like to thank the Swedish Foundation for Internationalization in
Higher Education and Research (STINT) for facilitating collabora-
tion between Sheffield and Uppsala.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201400335.
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
Scheme 1. Zinc complexes used in this study.
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position of the pyridyl ring (2), thus reflecting the steric
demands on the 2-amino group albeit with a minimal capacity
to provide hydrogen-bond donors. Modifying the substrate
binding pocket this way has also been suggested to provide
a hydrophobic cavity which could enhance electrostatic
interactions.[11] We were not able to oxidize the alcohol in 2.
This reaction always led to loss of the side chain, presumably
because of elimination reactions involving the central meth-
ylene group, and so we synthesized 4 by oxidizing 3. The
reaction we have studied is the cleavage of bis(p-nitrophenyl)
phosphate (BNPP) as a convenient model for DNA cleavage
under completely aqueous conditions at 258C, thus allowing
comparison of our data with that of previous reports.
ment in efficiency, and that it is likely to be a partial factor in
the 230-fold rate increase induced by using 2-amino substi-
tutions on the pyridyl ligands.
Similar first-order behavior is observed when 4 reacts with
BNPP at high pH, but at lower pH a nonlinear dependence on
concentration is apparent. We analyze this in terms of
relatively weak binding between the ligand and ZnII, as
confirmed by a potentiometric titration (see the Supporting
Information). At low pH, ligand protonation competes with
Zn complexation and the decrease in activity at low concen-
trations is due to the dissociation of Zn from the ligand.
Adding additional Zn ions increases the rate of the reaction,
thus showing a saturation curve with an apparent binding
constant which matches the parameters derived from the
titration data (see the Supporting Information), and leads to
a linear dependence on complex concentration. Plotting the
limiting second-rate constants for the reaction catalyzed by 4
at different pH values reveals a bell-shaped pH rate profile,
and the maximal activity of 4 is 70-fold greater than that for 1,
and 13-fold greater than that for 2. The bell-shape pH rate
profiles are fit to a reaction scheme where the singly
deprotonated species [Scheme 2 and Eq. (1)] is the kinetically
active ionic species.
The cleavage reaction shows a first-order dependence on
increasing complex concentration (0.2–1 mm) for 2, and the
pH dependence reveals a bell-shaped pH rate profile
(Figure 1). 31P NMR spectroscopy confirmed that 2 is phos-
Ka1½Hþꢀ
kp2H ¼ km2 ax½ZnLꢀ
À
Á
ð1Þ
Ka1Ka2 þ Ka1½Hþꢀ þ ½Hþꢀ½Hþꢀ
Figure 1. pH rate profile for the cleavage of BNPP catalyzed by 1 (no
symbols; from reference [12]), by 2 (triangles), by 3 (squares), and by
4 (circles) at 258C ([buffer]=0.05m). Solid lines are from fitting
Equation (1) to the data.
phorylated to produce a stable diester (no further reaction
over 20 days), and when excess substrate is used, only one
equivalent reacts with the complex. We note that the
introduction of a methyl group into the 2-position of the
pyridyl ligand provides a rate acceleration of about fivefold in
the maximum rate compared to the unsubstituted complex
1 (Table 1). This acceleration suggests that a steric effect
between substrate and ligand provides a moderate improve-
Scheme 2. Mechanistic scheme for reaction of 4 with BNPP.
In contrast to 2, when the reaction is monitored by
31P NMR spectroscopy, the observed product is 4-nitrophenyl
phosphate monoester, with no evidence of a transesterifica-
tion intermediate, and the complex completely hydrolyzes
5 equivalents of substrate (no substrate observable after
50 days), thus demonstrating catalytic turnover. As this
observation is also consistent with a metal-bound hydroxide
acting as the nucleophile, rather than the ligand, we tested 5
for activity: this complex showed no detectable activity under
the reaction conditions for over 46 days (see the Supporting
Information), and so we infer that 4 reacts through the
hydrated aldehyde nucleophile to form a transient intermedi-
ate which rapidly decomposes to form the original complex.
When 4 is recrystallized in the presence of methanol, the
hemiacetal 4’ is isolated and clearly shows that the zinc ion
Table 1: Collected kinetic parameters from pH rate profiles.
Compound
pKa1
pKa2
k2max [mꢁ1 sꢁ1
]
1[a] (H2O)
2 (H2O)
2 (MeOH)
3 (H2O)
3 (MeOH)
4 (H2O)
4’ (MeOH)
8.3[a]
10.9[a]
4.2ꢁ10ꢁ4[a]
7.50ꢂ0.03
8.2ꢂ0.2
7.9ꢂ0.1
9.5ꢂ0.1[b]
7.7ꢂ0.1
8.6ꢂ0.1
10.00ꢂ0.02
10.3ꢂ0.1
8.9ꢂ0.1
10.1ꢂ0.1[b]
8.4ꢂ0.1
9.7ꢂ0.1
2.3ꢂ0.04ꢁ10ꢁ3
4.7ꢂ0.6
1.3ꢂ0.2ꢁ10ꢁ2
23ꢂ5
2.9ꢂ0.3ꢁ10ꢁ2
9ꢂ1ꢁ10ꢁ2
[a] Reference [12]. [b] The pKa values are constrained to differ by 0.6
units, the closest that they can be without cooperative deprotonation
being required.[13]
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plausible. An estimate of bnuc ꢃ 1 can be made from their
data, and this predicts similar overall reactivity for both
tautomers when nucleophilicity and concentration are com-
bined. This value still predicts lower overall reactivity
compared to that of the more basic alkoxy nucleophiles
such as that in 3. However, we note that if the zinc is
coordinated by OH instead of Oꢁ, then presumably it will be
a more effective Lewis acid to activate the BNPP, and
combined with a different geometry for attack (involving
formation of a six-membered instead of four-membered ring),
this mechanism provides a plausible explanation as to why 4
has slightly higher maximal reactivity than 3.
It is not immediately obvious from the X-ray structure
(Figure 2) that the uncoordinated oxygen atom can act as
a nucleophile[15] towards a substrate coordinated to the Zn
ion, so we used computational methods to explore whether
more promising geometries are readily accessible. Starting
from the X-ray structure of 4’, we converted the methoxy
group into a hydroxy group, and the coordinated nitrate to
a water molecule, then minimized the structure using the
Hartree–Fock (HF) level of theory to optimize the geometry,
and DFT calculations to carry out single-point energy
calculations. The computational methodology used here is
at a similar level to that used by Ohanessian et al. for their
study of biomimetic Zn complexes.[16] In this study, it was
demonstrated that reliable results in terms of geometry and
chemical accuracy could be reached by simply performing
a HF geometry optimization, followed by a B3LYP energy
calculation with a larger basis set. In this work, we used the
M06-2X functional (instead of the popular B3LYP) as it also
includes dispersion correction (see the Supporting Informa-
tion for details). Changing the configuration of the hydrate
and reversing the propeller twist around the tertiary amine in
the complex revealed a structure that was essentially the same
in energy as the initial conformation (within 1 kcalmolꢁ1). By
introducing methyl 4-nitrophenyl phosphate to the Zn ion in
place of the water molecule, we find similar low-energy
conformations where the uncoordinated oxygen atom is
within 4.1 ꢀ of the phosphate, and close to in-line with the
leaving group (1648) as shown in Figure 2. Thus, the partic-
ipation of the noncoordinated oxygen atom as a nucleophile is
geometrically feasible. Performing a transition-state optimi-
zation for the nucleophile attack reaction of the noncoordi-
nated oxygen atom revealed a transition state which was
characterized by frequency calculations, and the minimum-
energy path connecting reactants to products through this
transition state was evaluated by calculating the intrinsic
reaction coordinate to confirm this is a viable pathway for the
phosphoryl transfer reaction (see the Supporting Information
for details).
Figure 2. a) Representation of the X-ray crystal structure of 4’ isolated
from methanol (hydrogen atoms and noncoordinated nitrate omitted
for clarity, except for OH coordinated to Zn). b) Optimized structure of
the monodeprotonated form of 4, with methyl 4-nitrophenyl phosphate
bound, at the HF/6-31+G*/LANL2DZ level of theory, using SMD
continuum solvent model (hydrogen atoms omitted for clarity, except
for OH coordinated to Zn).
can be coordinated by the hemiacetal form of the aldehyde
side chain, thus corroborating this interpretation (Figure 2).
The compound 3 behaves essentially the same way as 2
(undergoing a single reaction to produce a stable product),
but surprisingly is also significantly more reactive than 2
(sevenfold). There is no obvious explanation why methylating
the side chain should lead to a more active complex: the
Thorpe–Ingold effect usually enhances the formation of cyclic
compounds, but it seems more likely that perturbation of the
local solvation shell or indirect steric effects (e.g. with the
pyridyl groups) may affect the zinc coordination site and its
Lewis acidity.
As well as introducing turnover, the germinal diol
nucleophile has a lowered pKa value, thus bringing the
maximum activity closer to physiological pH. However,
Mancin and co-workers demonstrated that the cost of low-
ering the pKa value of zinc-coordinated alkoxides is to reduce
the activity of the nucleophile towards BNPP, and the overall
effect is a less reactive complex at all pH values, albeit with
a maximum closer to pH 7. Thus, the maximal reactivity of 4 is
expected to be some 65-fold lower than for 3, assuming that
the geminal hydroxy group has a similar effect on the OH
pKa value as a geminal trifluoromethyl group, rather than
twofold more reactive.[14]
This unexpected increase in activity leads us to question
whether the active nucleophile is coordinated to the zinc ion
(4 coord; Scheme 2) as has been generally assumed for these
type of metal ion complexes. In 4, the uncoordinated OH is an
alternative nucleophile, particularly if we consider the
tautomer where it is deprotonated (4 non-coord; Scheme 2)
as the reactive species. For this to be a viable possibility, the
bnuc for the reactions needs to be significant so that the greater
reactivity of the higher pKa anion can compensate adequately
for the unfavorable equilibrium between the tautomers. The
data of Mancin and co-workers[14] suggest that this is
A direct test of this proposal is not practical in aqueous
solution, as the two hydroxy groups cannot be distinguished.
If the reaction is carried out in dry methanol, the two sites are
differentiated as the noncoordinated position is methylated
(as illustrated by the crystal structure of 4’ in Figure 2). Thus,
we compared the reactivity in methanol solution (which is
known to provide a large rate acceleration for many zinc
complexes acting on phosphate esters).[17] Similarly to the
aqueous reactions, we observe a bell-shaped dependence on
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the most catalytically active tautomer will be the one in which
the noncoordinated hydroxy is ionized, thus furnishing a more
effective Lewis acid and more reactive nucleophile.
Experimental Section
Kinetic experiments were carried out at 258C, either in water with
50 mm buffer at 0.1m ionic strength (NaNO3) or in anhydrous
methanol with 50 mm buffer and monitored using UV/Vis spectros-
copy to measure the change in absorbance at 400 nm (water) or
320 nm (methanol). In water, a typical experiment was initiated by
the addition of 0.5 mL of 4 mm BNPP (in 50 mm buffer at 0.1m ionic
strength and 258C) to a 1 mL cuvette containing 0.5 mL of a solution
of Zn complex (in 50 mm buffer at 0.1m ionic strength) which had also
been equilibrated at 258C. In anhydrous methanol, 50 mL of 1 mm
BNPP (in anhydrous methanol) was added to a 1 mL cuvette
containing 0.95 mL of a solution of Zn complex (in 50 mm buffer)
and equilibrated at 258C. See the Supporting Information for details
of synthesis and characterization of ligands, kinetic and potentio-
metric data, product analyses, computational methods, and the CIF
for 4’.
Figure 3. pH rate profile for the cleavage of BNPP catalyzed by 2
(green triangles), by 3 (red squares), and by 4’ (blue circles) at 258C
in anhydrous methanol, [buffer]=0.05m). Solid lines are from fitting
Equation (1) to the data,[13] and the dashed lines illustrate the
corresponding reactivity in water.
Received: January 12, 2014
Revised: March 21, 2014
Published online: June 11, 2014
the pKa value, thus showing that the monodeprotonated
species is the dominant active form for 2, 3, and 4’
(Figure 3). Similar to the reports of Brown and co-workers,
we observe that in methanol, the rate of reaction of BNPP in
the presence of 2 and 3 is much greater, with increases in the
limiting second-order rate constants of about 1000-fold.[17]
However, the reactivity of 4’ is barely modified compared to
that of 4 (ca. threefold increase), and so in methanol the
maximal rate in the presence of 4’ is 300-fold slower than in
the presence of 3. We interpret this observation as confirming
the analysis of Mancin and co-workers, and that the activity of
the coordinated oxyanion is severely reduced because of the
inductive effect of the adjacent methoxy group.
Overall, the incorporation of an aldehyde functionality
has allowed conversion of a stoichiometric reagent into
a catalytic complex. The effect of methylating selected
positions provides approximately a tenfold enhancement in
the activity of the parent complex. Unexpectedly, the use of
an aldehyde hydrate as a nucleophile is not accompanied by
a decrease in reactivity relative to that of an alcohol, thus
leading us to propose that this system reacts through
a nucleophile which is not coordinated to the metal ion.
This proposal contrasts with the general strategy of designing
this type of complex, and suggests that incorporating non
metal-ion bound nucleophiles into a ligand may be a produc-
tive route to creating more effective complexes by avoiding
nucleophile deactivation through metal ion coordination, thus
enhancing the Lewis acidity of the metal ion in the active
tautomeric form, and allowing more favorable geometries for
the delivery of the nucleophile to the coordinated substrate.
We note that the mechanisms assumed for RNA coordinated
to metal ion complexes follow this mechanistic course (a
noncoordinated alkoxy nucleophile with a high pKa value), so
substituting the role of the 2’OH with a carbonyl hydrate site
on the ligand provides a strategy for designing complexes
effective for DNA hydrolysis as well. Finally, in considering
the active sites of sulfatases and phosphonohydrolases, which
use formyl glycine as a nucleophile, a metal ion is present and
coordinated to the hydrated aldehyde. Our data suggest that
Keywords: bioinorganic chemistry · DNA cleavage ·
.
enzyme models · kinetics · zinc
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