A highly reactive mononuclear Zn(II) complex for phosphodiester cleavage

Article abstract of DOI:10.1021/ja054003t

The activity of a Zn(II) complex of a tetradentate, tripodal ligand for catalyzing phosphodiester cleavage is enhanced 750-fold by introducing three hydrogen bond donors to the ligand. Inhibition studies show that the Zn-aqua complex is the kinetically ac

Full text of DOI:10.1021/ja054003t

Published on Web 09/08/2005
A Highly Reactive Mononuclear Zn(II) Complex for Phosphodiester Cleavage
Guoqiang Feng,† Juan C. Mareque-Rivas,*,‡ R. Torres Mart´ın de Rosales,‡ and
Nicholas H. Williams*,†
Centre for Chemical Biology, Department of Chemistry, UniVersity of Sheffield, Sheffield, United Kingdom S3 7HF,
and School of Chemistry, UniVersity of Edinburgh, Edinburgh, United Kingdom EH9 3JJ
Received June 16, 2005; E-mail: n.h.williams@sheffield.ac.uk; juan.mareque@ed.ac.uk
We report that the effect of introducing ligand-based hydrogen
bond donors to enhance the activity of a Zn(II) complex for
catalyzing phosphodiester cleavage can be dramatic. A detailed
mechanistic study shows that the effect is comparable to the rate
acceleration provided by the metal ion itself and generates a
mononuclear complex more active than the most reactive dinuclear
Zn(II) complex reported to date.
Enzymes and ribozymes that catalyze the hydrolysis of phosphate
esters frequently utilize metal ion cofactors in a central catalytic
role, and the most effective artificial systems have been based on
mimicking this activity using simple metal ion complexes. In return,
as it is easier to understand catalysis by small compared to large
molecules, establishing how these catalysts work provides insight
into the plausible catalytic roles of metal ions in biological catalysts.
In many cases, additional interactions in the active site supplement
Figure 1. pH rate profiles for the transesterification of 3: background
reaction (O, s-1); cleavage catalyzed by 1 (9, M-1 s-1); cleavage catalyzed
by 2 (b, M-1 s-1).
the effect of the metal ion center and influence the properties of
metal ion complexes,1 but there are only few reports of applying
this approach to the corresponding model complexes for phosphate
ester cleavage.2
Scheme 1
We have used the structurally homologous3,4 Zn(II) complexes
1 and 2 to examine the effect of introducing three aminopyridyl
hydrogen bond donors that are rigidly preorganized to interact with
a substrate coordinated to the Zn(II) ion. The reaction we selected
is the cleavage of 2-hydroxypropyl 4-nitrophenyl phosphate 3
(Ar ) 4-nitrophenyl), which undergoes intramolecular transesteri-
fication to produce propylene phosphate 4, and has been used as a
convenient model for RNA cleavage.
The cleavage reactions show a first-order dependence on
increasing complex concentrations (0.3-3 mM for 1, and 1-8 mM
for 2, with no indication of saturation) but are not accelerated by
doubling the buffer concentration and so are not subject to general
acid or base catalysis. In each case, the reaction catalyzed is the
cyclization to produce 4, and the complex undergoes multiple
turnovers as monitored by 31P NMR at pH 7. No hydrolysis of 4 is
observed over 7 days, and no reaction was observed when methyl
4-nitrophenyl phosphate, lacking an intramolecular nucleophile, was
used as the substrate. Figure 1 shows the pH dependence of the
cyclization of 3 in aqueous solution, and of the second-order rate
constant for catalysis by 1 and 2 (I ) 0.1 M (NaNO3), 50 mM
buffer at 25 °C). These reflect the concentration of the Zn-hydroxo
form of the free complex and nonlinear least-squares fitting of these
data yield kinetic pKa’s of 5.66 ( 0.05 (1) and 7.63 ( 0.05 (2), in
reasonable agreement with previously determined titration data.3
The limiting second-order rate constants are 7.9 ( 0.1 × 10-2 M-1
s-1 and 1.0 ( 0.1 × 10-3 M-1 s-1 for 1 and 2 respectively. The
background reaction is fit to specific base catalysis with a second-
order rate constant of 7.0 ( 0.4 × 10-2 M-1 s-1 (kb in Scheme 1,
blue).
To establish whether the differences observed are due to ground-
state or transition-state binding, the binding constants between the
substrate and phosphate esters must be evaluated. We estimated
this by measuring how effectively dimethyl phosphate (DMP) and
phenyl phosphate (PP) inhibit the cleavage reaction. Figure 2 shows
the normalized rate constants for the cleavage reaction catalyzed
by 1 (data for 2 not shown) with increasing inhibitor at different
pHs. These data are fit to eq 1, derived for competitive inhibition
† University of Sheffield.
‡ University of Edinburgh.
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13470
J. AM. CHEM. SOC. 2005, 127, 13470-13471
10.1021/ja054003t CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
substrate) concentrations are required to achieve the saturating
conditions to which they apply. An analogous analysis of the
dinuclear Zn(II) complex of Morrow and Richard,7 previously the
most effective Zn(II) complex reported to date for catalyzing the
transesterification of 3,8 leads to a rate acceleration of 1.6 × 105,
∼20-fold less effective than that of 1. The mononuclear control
gives a figure of 5.2 × 103, very similar to that of 2.
If we consider the rate acceleration under subsaturating condi-
tions, these figures become 3 × 108 M-1 for 1 and 3 × 104 M-1
for 2, a 104-fold difference;9 these accelerations are equivalent to
the formal dissociation constant of the transition state (KTS ) (kcat/
KM)/kuncat ≡ (kc/Kd)/kb in Scheme 1) from the catalyst.10 The catalytic
effect of 2 can be accounted for by considering the PP dianion as
a crude mimic of the dianionic intermediate which forms in the
rate-limiting step;11 the binding constant for PP agrees well with
Figure 2. Relative rate constants for the transesterification of 3 catalyzed
by 1 (0.3 mM) in the presence of phenyl phosphate (PP) and dimethyl
phosphate (DMP). O: DMP at pH 7.1; b: DMP at pH 6.1; 0: PP at pH
8.1; 9: PP at pH 7.1. The lines are curve fits to eq 1.
KTS and is presumably electrostatic stabilization of the developing
negative charge by the Zn ion. However, for 1 the same comparison
leaves a factor of (1.5 × 103)-fold which must be a consequence
of more specific stabilization of the transition state for cyclization,
presumably through enhanced hydrogen bonding between the ligand
amino groups and the transition state leading to the dianionic
intermediate.
Our results demonstrate that the benefits of tighter metal ion
coordination through tetradentate tripodal ligands can be retained
without sacrificing activity; in fact, this approach compares
favorably with the strategy of introducing a second metal ion center.
We are continuing our studies to characterize and exploit the effects
of these types of complexes in catalyzing phosphoryl transfer.
of the complex by inhibitor (I) where Kiobs is the observed
dissociation constant of I to the catalyst.
Kiobs
(Kiobs + [I])
k
k0
)
(1)
Significantly, Kiobs for both DMP and PP increases as the pH is
raised. If the Zn-hydroxo form of the complex is the catalytically
active form, which is the simplest interpretation of the pH rate
profile, Kiobs should be pH independent above the kinetic pKa.
Clearly, the esters inhibit the catalyzed reaction by competing for
the Zn-aqua form of the complex (Scheme 1, red). The mechanism
shown in Scheme 1 (black), where the substrate only binds to the
Zn-aqua form of the complex then undergoes specific base-catalyzed
transesterification,5 is consistent with all the data. This is kinetically
equivalent to catalysis by the Zn-hydroxo form of the complexes,
but mechanistically the limiting rate at high pH is explained by
deprotonation of the complex (Scheme 1, purple) reducing the
concentration of the active form in precise compensation for the
increasing hydroxide concentration.
Supporting Information Available: Derivation of equations and
procedures for curve fitting for inhibition data. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
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Commun. 2004, 610-611.
(5) Essentially the same scheme has been proposed for catalysis of 3′-uridyl
4-nitrophenyl phosphate cleavage by a dinuclear Zn(II) complex, based
on the lack of a significant solvent isotope effect. Yang, M.-Y.; Iranzo,
O.; Richard, J. P.; Morrow, J. R. J. Am. Chem. Soc. 2005, 127, 1064-
1065.
(6) A single aminopyridyl substituent has been shown to increase DMP
binding to a mononuclear Co(III) complex 33-fold. Chin, J.; Chung, S.;
Kim, D. H. J. Am. Chem. Soc. 2002, 124, 10948-10949.
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(8) Morrow, J. R.; Iranzo, O. Curr. Opin. Chem. Biol. 2004, 8, 192-200.
(9) Introducing two aminopyridyl substituents gives a 640-fold rate enhance-
ment over a parent Zn complex towards bis-4-nitrophenyl phosphate
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U.; Chin, J. Chem. Commun. 2004, 2862-2863.
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[H+]
([H+] + Ka)
Ki )
Kiobs
(2)
Combining eqs 1 and 2 (which shows the relation between Kiobs
,
Ka for the complex and the association constant, Ki, of the inhibitor
to the aqua form of the complex; full details available in Supporting
Information) gives Ki ) 10 ( 2 mM for 1 and 130 ( 30 mM for
2 for the DMP anion,6 and of 5 ( 1 µM for 1 and 60 ( 20 µM for
2 for the PP dianion.4
The rate acceleration achieved by coordinating 3 to 1 or 2 is
given by the ratio of kc to kb (i.e. when the catalyst is saturated
with substrate); assuming that the binding constant for DMP is a
good approximation for substrate binding (Kd in Scheme 1), kc )
2 × 105 M-1 s-1 (1) and 3 × 102 M-1 s-1 (2), and thus the
cyclization of 3 is accelerated (3 × 106)-fold by 1, and (4 × 103)-
fold by 2. Remarkably, the enhancement brought about by the three
amino groups provides a contribution to catalysis comparable to
the core Zn(II) ion in 2, giving an additional 750-fold acceleration
in the catalyst-substrate complex. These rate-limiting accelerations
are constant, but above the kinetic pKa increasing catalyst (or
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