Substrate specificity of an active dinuclear Zn(II) catalyst for cleavage of RNA analogues and a dinucleoside

Article abstract of DOI:10.1021/ja056167f

The cleavage of the diribonucleoside UpU (uridylyl-3′-5′- uridine) to form uridine and uridine (2′,3′)-cyclic phosphate catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)- 2-hydroxypropane (Zn₂(1)(H₂O)) has been studied at pH 7-10 and 25 °C. The kinetic data are consistent with the accumulation of a complex between catalyst and substrate and were analyzed to give values of k_c (S^-1), K_d (M), and k_c/K _d (M^-1 s^-1) for the Zn₂(1)(H ₂O)-catalyzed reaction. The pH rate profile of values for log k _C/Kd for Zn₂(1)(H₂O)-catalyzed cleavage of UpU shows the same downward break centered at pH 7.8 as was observed in studies of catalysis of cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) and uridine-3′-4-nitrophenyl phosphate (UpPNP). At low pH, where the rate acceleration for the catalyzed reaction is largest, the stabilizing interaction between Zn₂(1)(H₂O) and the bound transition states is 9.3, 7.2, and 9.6 kcal/mol for the catalyzed reactions of UpU, UpPNP, and HpPNP, respectively. The larger transition-state stabilization for Zn ₂(1)(H₂O)-catalyzed cleavage of UpU (9.3 kcal/mol) compared with UpPNP (7.2 kcal/mol) provides evidence that the transition state for the former reaction is stabilized by interactions between the catalyst and the C-5′-oxyanion of the basic alkoxy leaving group.

Full text of DOI:10.1021/ja056167f

Published on Web 01/13/2006
Substrate Specificity of an Active Dinuclear Zn(II) Catalyst for
Cleavage of RNA Analogues and a Dinucleoside
AnnMarie O’Donoghue,† Sang Yong Pyun,‡ Meng-Yin Yang,†
Janet R. Morrow,*,† and John P. Richard*,†
Contribution from the Departments of Chemistry, UniVersity at Buffalo, State UniVersity of New
York, Buffalo, New York 14260-3000 and Pukyong National UniVersity, Busan 608-737, Korea
Received September 7, 2005; E-mail: jmorrow@buffalo.edu; jrichard@buffalo.edu
Abstract: The cleavage of the diribonucleoside UpU (uridylyl-3′-5′-uridine) to form uridine and uridine (2′,3′)-
cyclic phosphate catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-
hydroxypropane (Zn2(1)(H2O)) has been studied at pH 7-10 and 25 °C. The kinetic data are consistent
with the accumulation of a complex between catalyst and substrate and were analyzed to give values of
kc (s-1), Kd (M), and kc/Kd (M-1 s-1) for the Zn2(1)(H2O)-catalyzed reaction. The pH rate profile of values for
log kc/Kd for Zn2(1)(H2O)-catalyzed cleavage of UpU shows the same downward break centered at pH 7.8
as was observed in studies of catalysis of cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP)
and uridine-3′-4-nitrophenyl phosphate (UpPNP). At low pH, where the rate acceleration for the catalyzed
reaction is largest, the stabilizing interaction between Zn2(1)(H2O) and the bound transition states is 9.3,
7.2, and 9.6 kcal/mol for the catalyzed reactions of UpU, UpPNP, and HpPNP, respectively. The larger
transition-state stabilization for Zn2(1)(H2O)-catalyzed cleavage of UpU (9.3 kcal/mol) compared with UpPNP
(7.2 kcal/mol) provides evidence that the transition state for the former reaction is stabilized by interactions
between the catalyst and the C-5′-oxyanion of the basic alkoxy leaving group.
Introduction
complementarity to the transition state for the catalyzed reaction.
On the other hand, it is early to conclude that the limits are
In recent years attempts to design and synthesize low
molecular weight catalysts with activities that approach those
of protein and RNA catalysts have produced many significant
and interesting results1-17 but no major breakthroughs. This may
be a sign that large molecular weights are somehow essential
to the construction of catalysts that show a high degree of
being approached for the activity of small molecule catalysts.
We have shown that the small molecule dinuclear zinc
complex Zn2(1)(H2O) gives a transition-state stabilization for
catalysis of cleavage of the simple phosphodiester 2-hydroxy-
propyl-4-nitrophenyl phosphate (HpPNP) that is a substantial
fraction (e50%) of the maximum possible for a hypothetical
protein or ribozyme catalyst of the same reaction.18,19 Zn2(1)-
(H2O) owes its high activity to cooperative interactions between
the tethered Zn(II) cations and the linker alkoxide anion, which
draw the cations into a densely charged core that interacts
effectively with the anionic transition state for phosphodiester
cleavage.19-23 We want to extend this characterization of the
unusual catalytic power of Zn2(1)(H2O) to the cleavage of
ribonucleic acids in order to obtain insight into the origin of
the rate accelerations for metalloenzyme-24 and ribozyme-
catalyzed25,26 cleavage of RNA and because a better understand-
ing of what limits the catalytic power of Zn2(1)(H2O) will help
† State University of New York, Buffalo.
‡ Pukyong National University.
(1) Wall, M.; Hines, R.; Chin, J. Angew. Chem., Int. Ed. Engl. 1993, 32, 1633-
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9
10.1021/ja056167f CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 1615-1621
1615

A R T I C L E S
Scheme 1
O’Donoghue et al.
cleavage of UpU, where the leaving group is strongly basic,
than of the transition state for the cleavage of UpPNP, where
the leaving group is the weakly basic 4-nitrophenoxide ion. This
provides evidence for a modest stabilization of negative charge
at the leaving group of UpU by interaction with Zn2(1)(H2O).
We also present a comparison of the specificity of Zn2(1)(H2O)
for catalysis of the cleavage reactions of HpPNP, UpPNP, and
UpU as measured by the net stabilization by the catalyst of the
transition state for the uncatalyzed reaction, which provides
insight into the strengths and limitations of Zn2(1)(H2O) as a
catalyst of phosphate diester cleavage.
Experimental Section
+
All buffers, Zn(NO3)2, uridylyl(3′,5′)uridine (UpU, NH4 salt),
uridine (2′,3′)-cyclic monophosphate (2′,3′-cUMP), uridine-3′-mono-
phosphate (3′-UMP), uridine 2′-monophosphate (2′-UMP), uridine, and
uracil were reagent grade from Sigma-Aldrich. Water was distilled and
then passed through a Milli-Q purification system. The ligand 1 was
prepared by literature procedures.20,33,34 A stock solution of UpU was
shown to be stable to cleavage for a period of several weeks at neutral
pH. The solution pH was determined at 25 °C using an Orion digital
pH meter equipped with a temperature compensation probe. Procedures
described in earlier work were used in preparing solutions of Zn2(1)-
(H2O) for kinetic studies.19
to guide the rational incorporation of design elements to
overcome these limits.
Chromophoric substrates were used in earlier studies on
Zn2(1)(H2O) because of the ease of monitoring reactions by
UV spectroscopy.19-23 The 4-nitrophenoxide ion leaving group
of HpPNP and uridine-3′-4-nitrophenyl phosphate (UpPNP)
is weakly basic and intramolecular addition of the C-2′ ribosyl
oxyanion to the substrate phosphate must be rate determining
if the reactions proceed by a stepwise mechanism through an
oxyphosphorane dianion reaction intermediate.27 RNA differs
from HpPNP and UpPNP in that the C-5′ alkoxy oxygen of
the nucleoside leaving group is strongly basic. Therefore,
breakdown of the intermediate with expulsion of the leaving
group anion will be the rate-determining step, if the reaction
mechanism is stepwise, or there will be partial cleavage of the
bond to the leaving group and development of negative charge
at oxygen, if the reaction mechanism is concerted.26-31 In either
case stabilization of negative charge at this leaving oxygen at
the rate-determining transition state might contribute to the rate
acceleration for Zn2(1)(H2O)-catalyzed cleavage of RNA but
not for the catalyzed reactions of HpPNP and UpPNP.32 We
therefore extended our studies on Zn2(1)(H2O) to the ribodi-
nucleotide substrate UpU in which the leaving group is the same
as for RNA to determine if an enhanced rate acceleration is
observed for this substrate that is consistent with a stabilizing
electrostatic interaction between the catalyst and the strongly
basic anionic leaving group
HPLC Product Analysis. The cleavage of UpU was monitored by
separating the reactant and products over a C18 radial-pak column (250
mm × 4.6 mm) using a Waters 600E HPLC equipped with a 490 UV-
Vis detector with peak detection at 260 nm. An isocratic elution solvent,
prepared by mixing nine parts water at pH 4.3 (60 mM acetic acid
buffer and 0.10 M NH4Cl) with one part methanol, was used for all
separations. The sodium salt of p-nitrobenzenesulfonate was included
with all samples as an internal standard to allow for corrections in small
variations in the volume of the sample injected. The reactant UpU and
products 2′,3′-cUMP, 3′-UMP, 2′-UMP, and uridine were identified
by comparison of retention times with those of authentic material. The
extinction coefficients at 260 nm, relative to a standard value of 1.0
for uracil, were determined from the ratio of concentrations and peak
areas from HPLC analysis of known concentrations of the following
compounds: uracil, 1.00; 2′,3′-cUMP, 1.06; 3′-UMP, 0.91; 2′-UMP,
0.96; uridine, 1.19; UpU, 2.32.
Kinetic Measurements. The following buffers were used for these
experiments: imidazole, pH 7.0-7.4; 2-(N-cyclohexylamino)ethane-
sulfonic acid (CHES), pH 8.9-9.3; 3-(cyclohexylamino)-1-propane-
sulfonic acid (CAPS), pH 9.8. The solutions used for kinetic analyses
were prepared by mixing measured amounts of UpU, Zn2(1)(H2O),
,p-nitrobenzene-sulfonate ion and the appropriate buffer to give final
concentrations of 0.070 mM UpU, 0-2.00 mM Zn2(1)(H2O), 0.020
mM p-nitrobenzenesulfonate ion, and 20 mM buffer at I ) 0.10
(NaNO3) in a volume of 0.50 mL. The solution was kept at 25 °C; at
measured times 50 µL aliquots were withdrawn, the pH adjusted to
4-5 with a small volume of acetate buffer; the relative concentrations
of substrate UpU and products were determined by HPLC analysis.
First-order rate constants kobsd (s-1) were determined as the slopes of
linear plots of reaction progress against time over the first 2-8%
reaction using eq 1, where [Uridine] is the concentration of the cleavage
product and [UpU]o is the initial concentration of the substrate. Rate
constants for the solvent-catalyzed reaction were determined under the
same conditions but in the absence of Zn2(1)(H2O). Linear and
nonlinear least-squares fits of kinetic data to the appropriate kinetic
equations were performed using SigmaPlot from Jandel Scientific.
We report here kinetic parameters over a broad range of pH
for the cleavage of UpU (Scheme 1) catalyzed by Zn2(1)(H2O)
and a comparison of these data with the corresponding
parameters for Zn2(1)(H2O)-catalyzed cleavage of HpPNP19
and UpPNP.21 Our data show that Zn2(1)(H2O) provides 2.1
kcal/mol greater stabilization of the transition state for the
(27) Perreault, D. M.; Anslyn, E. V. Angew. Chem., Int. Ed. Engl. 1997, 36,
432-450.
(28) Skoog, M. T.; Jencks, W. P. J. Am. Chem. Soc. 1984, 106, 7597-7606.
(29) Stefanidis, D.; Cho, S.; Dhe-Paganon, S.; Jencks, W. P. J. Am. Chem. Soc.
1993, 115, 1650-1656.
(30) Lonnberg, H.; Stromberg, R.; Williams, A. Org. Biomol. Chem. 2004, 2,
2165-2167.
(31) Davis, A. M.; Hall, A. D.; Williams, A. J. Am. Chem. Soc. 1988, 110,
5105-5108.
[Uridine]
) kobsd
t
(1)
[UpU]o
(32) Menger, F. M.; Ladika, M. J. Am. Chem. Soc. 1987, 109, 3145-3146.
9
1616 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Zn(II) Catalyst for Cleavage of RNA Analogues
A R T I C L E S
Figure 1. Increase in the fractional conversion of UpU to uridine (fU) with
time for Zn2(1)(H2O)-catalyzed reactions at 25 °C. The slope of these linear
correlations is equal to kobsd (s-1) for the Zn2(1)(H2O)-catalyzed reaction.
(A) Reactions at pH 7.0: (b) 0.5 mM, (1) 1.0 mM, (9) 1.5 mM, (2) 2.0
mM [Zn2(1)(H2O)]T. (B) Reactions at pH 9.5: (b) 0.25 mM, (1) 0.50
mM, (9) 1.5 mM, (2) 2.0 mM Zn2(1)(H2O).
Figure 2. Effect of increasing concentrations of catalyst Zn2(1)(H2O) on
the observed first-order rate constant kobsd for cleavage of UpU at 25 °C.
The solid lines show the nonlinear least-squares fit of the experimental data
to eq 2 derived for Scheme 2. Reactions at pH 7.0 (b), 7.4 (9), 8.9 (2),
9.3 ([), 9.5 (O), 9.8 (0).
Scheme 2
Results
The cleavage of UpU (0.070 mM) catalyzed by Zn2(1)(H2O)
at 25 °C and pH 7.0-9.8 maintained with 20 mM of the
appropriate buffer at I ) 0.10 M (NaNO3) was monitored by
HPLC. These analyses showed that this reaction gives uridine
and 2′,3′-cUMP as products at early times and that 2′,3′-cUMP
undergoes hydrolysis to 3′-UMP and 2′-UMP at longer times.
Table 1. Kinetic Parameters Kd, kc, and (kc/Kd)app ) (kZn
)
for
app
Zn2(1)(H2O)-Catalyzed Cleavage of UpU at 25 °C
1
1 c,d
pH
(kZn)
aM-1 s-
Kd/mMb,d
kc/10-6 s-
app
The kinetic parameters for the cleavage of UpU catalyzed
by Zn2(1)(H2O) were determined by monitoring the disappear-
ance of UpU and the formation of uridine by HPLC. Figure 1
shows representative plots of the fractional conversion of UpU
to uridine (fU) with time during the initial stages of reactions at
pH 7.0 and 9.5 (25 °C). The different solid lines show data for
reactions catalyzed by different [Zn2(1)(H2O)]T , where [Zn2-
(1)(H2O)]T is the sum of the concentrations of the catalyst in
the protonated ([Zn2(1)(H2O)]) and ionized ([Zn2(1)(HO-)])
forms (see below). Values of kobsd for the reactions of UpU
were calculated from the slopes of these linear correlations
(eq 1).
7.0
7.4
8.9
9.3
9.5
9.8
0.00056
0.00099
0.0034
0.0035
0.0045
0.0068
3.5 ( 0.5
2.4 ( 0.9
0.80 ( 0.13
1.9 ( 0.3
1.9 ( 0.3
1.2 (0.01
2.0 ( 0.2
2.4 ( 0.6
2.8 ( 0.2
6.4 ( 0.3
8.4 ( 1.0
7.9 ( 0.6
a Apparent second-order rate constant for the Zn2(1)(H2O)-catalyzed
reaction, calculated as the ratio kc/Kd. b Dissociation constant of the substrate
for Zn2(1)(H2O) calculated from the nonlinear least-squares fit of the data
from Figure 2 to eq 2 derived for Scheme 2. c Limiting rate constant at
high concentrations of UpU calculated from the nonlinear least-squares fit
of the data from Figure 2 to eq 2 derived for Scheme 2. d The uncertainties
in these kinetic parameters are from the nonlinear least-squares analysis of
the data.
(g0.1 mM) used in these experiments as was assumed in the
derivation of eq 2.
kc[Zn2(1)(H2O)]T
kobsd
)
(2)
[
]
Linear plots of kobsd against the catalyst concentration [e2
mM] were observed for Zn2(1)(H2O)-catalyzed cleavage of the
chromophoric substrate UpPNP at pH 7.0.21 Figure 3 shows
that there is a small downward curvature in the plot of kobsd
against [Zn2(1)(H2O)] for cleavage of UpPNP as the concentra-
tion of the catalyst is increased to 6.0 mM. The solid line through
the experimental data in Figure 3 shows the fit from nonlinear
least-squares analysis of the experimental data to eq 2 derived
Kd + [Zn2(1)(H2O)]T
Figure 2 shows the increase in kobsd for the cleavage of UpU
as [Zn2(1)(H2O)]T is increased from 0 to 2.0 mM for reactions
at 25 °C, pH 7.0-9.8, 20 mM buffer, and I ) 0.10 (NaNO3).
The downward curvature observed for these plots is consistent
with the formation of a complex between substrate UpU and
catalyst Zn2(1)(H2O). The solid lines through the experimental
data in Figure 2 show the fit from nonlinear least-squares
analysis of the experimental data to eq 2 derived for Scheme 2.
Table 1 lists the kinetic parameters kc (s-1), Kd (M), and (kc/
Kd)app (M-1 s-1) determined by this fitting procedure. The values
of Kd (Table 1) are >10-fold larger than [UpU] ) 0.070 mM
(Table 1). Under these conditions >90% of the catalyst will be
in the free form ([[Zn2(1)(H2O)]T] . [[Zn2(1)(H2O)]T‚UpX],
Scheme 2) for reactions at all concentrations of Zn2(1)(H2O)
for Scheme 2 using values of Kd ) 9 mM and kc ) 0.31 s-1
.
Apparent second-order rate constants (kZn)app (M-1 s-1) for
Zn2(1)(H2O)-catalyzed cleavage of UpU were determined as
(kZn)app ) (kc/Kd)app (Table 1). These second-order rate constants
have the same physical significance as second-order rate
constants (kZn)app (M-1 s-1) reported in earlier studies of
Zn2(1)(H2O)-catalyzed cleavage of UpPNP21 and HpP-
NP,19,20,22 which were determined as the slopes of linear plots
of kobsd against [Zn2(1)(H2O)]T. Figure 4 shows a plot of log-
(kc/Kd)app ) (kZn)app for Zn2(1)(H2O)-catalyzed cleavage of UpU
and the plot of log(kZn)app against pH for Zn2(1)(H2O)-catalyzed
(33) McCue, D. P. Ph.D. Thesis, University at Buffalo, SUNY: Buffalo, 1999.
(34) Atkins, T. J.; Richman, J. E.; Oettle, W. F. Org. Synth. 1978, 58, 86-98.
9
J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006 1617

A R T I C L E S
O’Donoghue et al.
Scheme 3
concentration of hydroxide ion. The solid line through the
experimental data shows the fits of the experimental data
obtained using values of kHO ) 0.0011 and 1600 M-1 s-1
,
respectively, for hydroxide-ion-catalyzed cleavage of UpU and
UpPNP determined by nonlinear least-squares analyses.
Discussion
The cleavage of UpU and related diribonucleosides is very
slow at room temperature, and these reactions are therefore
normally studied at elevated temperature.35-38 The efficient
catalysis of cleavage of UpU by Zn2(1)(H2O) allows us to study
this reaction at the same temperature (25 °C) used for earlier
studies on Zn2(1)(H2O)-catalyzed cleavage of HpPNP19 and
UpPNP.21 The apparent second-order rate constants log(kc/Kd)app
) (kZn)app for Zn2(1)(H2O)-catalyzed cleavage of UpU are first
order in hydroxide ion at neutral pH and show a downward
break centered at pH ) pKa ) 7.8 for ionization of Zn2(1)-
(H2O) to Zn2(1)(HO-). The same pH dependence was reported
in earlier work for Zn2(1)(H2O)-catalyzed reactions of HpPNP19
and UpPNP.21 This shows that each of these three catalyzed
reactions proceeds by reaction of the same ionic forms of
substrate and catalyst.23 The data in Table 1 suggest that there
is both a small decrease in Kd and an increase in kc with
increasing pH; however, the scatter in the data is large. These
changes provide evidence that both the ground-state complex
between substrate and catalyst and the transition state for
conversion of this complex to products are stabilized at
increasing pH. The discussion in this paper will focus on the
larger changes in (kZn)app ) (kc/Kd)app for which a good
correlation with changing pH is observed.
Figure 3. Effect of increasing concentrations of catalyst Zn2(1)(H2O) on
the observed first-order rate constant kobsd for cleavage of UpPNP for
reactions at 25 °C and pH 7.0. The solid lines show the nonlinear least-
squares fit of the experimental data to eq 2 derived for Scheme 2.
Figure 4. pH rate profiles of second-order rate constants kHO and (kZn
)
app
Substrate Specificity. The large rate accelerations achieved
by enzyme catalysts are due to the high degree of complemen-
tarity between the protein and the transition state for the
catalyzed reaction. This generally results in a high specificity
of the protein for binding to the transition state.39-41 Zn2(1)-
(H2O) is a very effective catalyst for the cleavage of HpPNP
and achieves ≈50% of the reduction in the activation barrier
for the uncatalyzed reaction (transition-state stabilization) that
would be observed for optimal enzymatic catalysis of this
reaction.19 Small molecule catalysts such as Zn2(1)(H2O) do
not have the well-defined binding pocket required to show a
high degree of complementarity for the transition state for the
cleavage of HpPNP or cleavage of any other phosphate diester.
However, the tight binding of Zn2(1)(H2O) to the transition state
for phosphodiester cleavage reflects in some sense the specificity
of the catalyst for transition-state binding, and this specificity
can be determined through an examination of the transition-
state stabilization for catalysis of reactions of different substrates.
One measure of substrate specificity of Zn2(1)(H2O) is the
binding energy observed upon transfer of the transition state of
the uncatalyzed reaction from solution to the catalyst.39,41
for cleavage of phosphodiester substrates UpPNP and UpU catalyzed by
hydroxide ion and Zn2(1)(H2O). UpPNP [data from ref 21]: ([) kHO, (1)
(kZn)app. UpU: (2) kHO, (b) (kZn)app. The solid lines through values for
(kZn
)
show the theoretical fits of the data to eq 3 derived for Scheme 3.
app
cleavage of UpPNP taken from earlier work.21 The solid lines
through these two sets of experimental data were drawn using
eq 3 derived for Scheme 3 and values of kZn ) 0.005 and 200
M-1s-1, respectively, for Zn2(1)(H2O)-catalyzed cleavage of
UpU and UpPNP21 and a pKa of 7.8.21,22 The value of pKa )
7.8 determined from the nonlinear least-squares fit of kinetic
data for the reactions of UpPNP21 and HpPNP19 is in agreement
with the value of pKa ) 8.0 determined by potentiometric
titration.19 The value of (kZn)app ) (kc/Kd)app ) 34 M-1 s-1 for
Zn2(1)(H2O)-catalyzed cleavage of UpPNP at pH ) 7.0
calculated from the kinetic parameters determined for Figure 3
is in agreement ((10%) with (kZn)app ) 27 M-1 s-1 that can be
calculated from eq 3 using 200 M-1 s-1 and Ka ) 10-7.8 M.21
kZnKa
(kZn)app ) (kc/Kd)app
)
(3)
+
[
]
Ka + [H ]
Figure 4 also shows the pH rate profiles of the observed first-
order rate constants for spontaneous cleavage of UpU and
UpPNP.21 Both cleavage reactions are first order in the
(35) Anslyn, E.; Breslow, R. J. Am. Chem. Soc. 1989, 111, 4473-4482.
(36) Kirby, A. J.; Marriott, R. E. J. Am. Chem. Soc. 1995, 117, 833-834.
(37) Kiviniemi, A.; Lonnberg, T.; Ora, M. J. Am. Chem. Soc. 2004, 126, 11040-
11045.
9
1618 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Zn(II) Catalyst for Cleavage of RNA Analogues
A R T I C L E S
Scheme 4
kZnKa/Kw
∆GS† ) ∆GE† - ∆GN† ) -RT ln
(4)
[
]
kHO
Table 2. Kinetic Parameters for Zn2(1)(H2O)- and
Hydroxide-Ion-Catalyzed Cleavage of HpPNP, UpPNP, and UpU
at 25 °C
HpPNPa
UpPNPb
UpUc
d
k
Zn (M-1 s-1
)
0.71
200
0.005
(kZnKa/Kw) (M-2 s-1
)
1.1 × 106
3.2 × 108
8 × 103
0.0011
7 × 106
e
f
k
HO (M-1 s-1
)
0.099
1600
(kZnKa/Kw)/kHO (M-1
)
1.1 × 107
2.0 × 105
g
†
(∆GS
)
max
h (kcal/mol)
-9.6
-7.2
-9.3
Figure 5. Comparison of the estimated changes in Gibbs free energy for
formation of ground-state (∆GES) and transition-state (∆GS†) complexes
for the Zn2(1)(H2O)-catalyzed reactions of HpPNP, UpPNP, and UpU.
a Data from ref 19. b Data from ref 21. c This work. d Limiting second-
order rate constant observed for the Zn2(1)(H2O)-catalyzed reaction at high
pH. e Apparent third-order rate constant calculated for the Zn2(1)(H2O)-
catalyzed reaction at low pH. f Apparent second-order rate constant for the
hydroxide-ion-catalyzed reaction. g Rate acceleration for 1.0 M Zn2(1)(H2O)
at low pH where the apparent rate constant for the Zn2(1)(H2O)-catalyzed
reaction is first order in [HO-]. h Stabilization of the transition state for
the solution reaction at low pH by interaction with 1.0 M Zn2(1)(H2O).
(1) Brønsted Catalysis. The observation of a small inverse
solvent deuterium isotope effect on Zn2(1)(H2O)-catalyzed
cleavage of UpPNP shows that there is no formal proton transfer
from the C-2 hydroxyl of substrate to the catalyst at the transition
state for this reaction.23 This provides strong evidence that
formal Brønsted acid-base catalysis is not significant for Zn2-
(1)(H2O)-catalyzed reactions when formation of an oxyphos-
phorane intermediate or oxyphosphorane-like transition state is
rate determining.23 It is unlikely that formal Brønsted acid-
base catalysis is significant for the cleavage of UpU, where
breakdown of an oxyphosphorane intermediate or oxyphospho-
rane-like transition state is rate determining. This is because
similar mechanisms are expected for formation and breakdown
of this intermediate/transition state in reactions that are nearly
the microscopic reverse of one another. We suggested that
Brønsted acid-base catalysis by Zn2(1)(H2O) is not favored
because transition-state stabilization is largely due to the
electrostatic interaction between the cationic catalyst and
oxyphosphorane dianion.23 A significant net stabilization of the
transition state for phosphodiester cleavage by proton transfer
to the nucleophile/leaving group anion is unlikely for small
molecular metal ion catalysts such as by Zn2(1)(H2O) because
such proton transfer will cause a decrease in negative charge at
the nucleophile/leaving group and in the stabilizing electrostatic
interaction between catalyst and the transition state.
Equation 4 derived for Scheme 4 shows that this binding energy
can be calculated from the ratio of the rate constants for (1) the
apparent third-order rate constant for the Zn2(1)(H2O)-catalyzed
reaction at low pH, where the catalyst exists mainly in the
protonated form (kZnKa/Kw) and where the catalyzed reaction
is first-order in [HO-] (Figure 4); this rate constant was
calculated from eq 3 when [H+] . Ka and using [H+] ) Kw/
[HO-]; and (2) the second-order rate constant kHO for the
reaction catalyzed by hydroxide ion.
Table 2 lists the rate constants determined for the spontaneous
and Zn2(1)(H2O)-catalyzed reactions of HpPNP, UpPNP, and
UpU and the transition-state binding energies ∆Gs† that have
been calculated from these rate constants using eq 4. These
transition-state binding energies are summarized in Figure 5.
Transition-State Binding Energy. There are at least three
interactions between transition state and catalyst that may
contribute to the stabilization of the transition state for Zn2(1)-
(H2O)-catalyzed cleavage of phosphate diesters: (1) Brønsted
acid-base catalysis by proton transfer at the attacking nucleo-
philic oxygen of HpPNP or UpPNP when formation of an
oxyphosphorane dianion intermediate is rate determining or to
the leaving group oxygen of UpU when breakdown of the
intermediate is rate determining;27 (2) Interactions between the
catalyst and the uridine nucleobase of UpPNP or UpU;42-44
(3) Electrostatic interactions between the oppositely charged
substrate and catalyst. We consider now the relative importance
of these interactions in Zn2(1)(H2O)-catalyzed cleavage of
HpPNP, UpPNP, and UpU.
(2) Interactions with Uracil Groups. There is a correlation
between the number of uracil groups at the phosphate monoan-
ion substrate and the binding affinity for Zn2(1)(H2O). The value
of Ki ) Kd ) 16 mM determined for binding of the minimal
ligand dimethyl phosphate to Zn2(1)(H2O) at pH 7.6 (∆GES
)
-2.4 kcal/mol, Figure 5)19,45 decreases to Kd ) 9 mM (∆GES
) -2.8 kcal/mol) and 3.5 mM (∆GES ) -3.3 kcal/mol),
respectively, for Zn2(1)(H2O)-catalyzed cleavage of UpPNP
and UpU at pH 7.0. The increased stabilization of the complex
between catalyst and substrate with increasing uracil groups
suggests that this complex is stabilized by interactions between
(38) Mikkola, S.; Stemman, E.; Nurmi, K.; Yousefi-Salakdeh, E.; Stromberg,
R.; Lonnberg, H. J. Chem. Soc., Perkin Trans. 2 1999, 1619-1625.
(39) Wolfenden, R. Nature 1969, 223, 704-705.
(40) Pauling, L. Nature 1948, 161, 707-709.
(41) Lienhard, G. E. Science 1973, 180, 149-154.
(42) Shionoya, M.; Kimura, E.; Shirot, M. J. Am. Chem. Soc. 1993, 115, 6730-
6737.
(43) Aoki, S.; Kimura, E. J. Am. Chem. Soc. 2000, 122, 4542-4548.
(44) Kimura, E.; Kitamura, H.; Ohtani, K.; Koike, T. J. Am. Chem. Soc. 2000,
122, 4668-4677.
(45) A smaller apparent inhibition constant of 11 mM for diethyl phosphate
will be observed at pH 7.0 if this inhibitor binds specifically to the
protonated form of the catalyst.
9
J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006 1619

A R T I C L E S
Scheme 5
O’Donoghue et al.
ligands whose charge increases from -1 at the Michaelis
complex to -2 at the transition state for phosphate diester
cleavage.
(2) There is 2.4 kcal/mol greater stabilization of the transition
state for cleavage of the minimal substrate HpPNP by interac-
tion with Zn2(1)(H2O) than of the transition state for cleavage
of UpPNP (Figure 5).21 This is consistent with the notion that
access of the more bulky substrate UpPNP to the charged
cationic core of Zn2(1)(H2O) is restricted and that the small
substrate HpPNP may approach more closely, allowing for
stronger electrostatic stabilization of the anionic transition state.
the catalyst and N-3 of the pyrimidine ring, as has been reported
for analogous Zn(II) complexes.42-44 However, these interac-
tions are weak, and there is insufficient data to define their site
at the pyrimidine ring.
(3) The observation that the rate-determining transition state
for cleavage of UpU is 2.1 kcal/mol more strongly stabilized
(tightly bound) by Zn2(1)(H2O) than the transition state for
cleavage of UpPNP (Figure 5) provides evidence that the
stabilizing interaction between the catalyst and the C-5′ leaving
group oxyanion at the transition state for cleavage of UpU is
stronger than the corresponding interactions between the catalyst
and the nucleophilic C-2′ oxyanion at the transition state for
cleavage of UpPNP. This conclusion will hold for either a
stepwise mechanism and a pentavalent oxyphosphorane transi-
tion state or a concerted mechanism.26,27,30,31 In either case, the
barrier to expulsion of the leaving group anion will make the
largest contribution to the barrier for the uncatalyzed cleavage
of the substrate UpU with the strongly basic alkoxide ion leaving
group, so that this site will provide a target at UpU for
interaction with the cationic catalyst.
On the other hand, there is no simple correlation between
the magnitude of stabilization of the ground-state complexes
to Zn2(1)(H2O) and of the transition state for the catalyzed
cleavage reactions (Figure 5). The largest transition-state
stabilization is observed for the minimal substrate HpPNP
(∆GS† ) 9.6 kcal/mol, Table 2) that binds most weakly to
Zn2(1)(H2O). A weaker transition stabilization of ∆GS† ) 7.2
kcal/mol is observed for UpPNP, a substrate with a single uracil
group which apparently interacts with the catalyst in the ground-
state complex. Finally, the 2.1 kcal/mol larger transition-state
stabilization observed for the catalyzed reaction of UpU (∆GS†
) 9.3 kcal/mol) compared to UpPNP (∆GS† ) 7.2 kcal/mol)
is greater than the 0.5 kcal/mol difference in the stabilization
of ground-state complexes to the catalyst.
This difference in the effect of changing substrate structure
on the ground and transition-state binding energy provides
evidence that the interactions between the pyrimidine ring(s)
of substrate and catalyst are expressed upon formation of a
nonproductive complex between substrate and catalyst (Kd′,
Scheme 5) but weaken at the productive catalytic complex (Kd,
Scheme 5).46 Such nonproductive binding will cause an apparent
stabilization of the Michaelis complex and a reduction in the
concentration of the catalytically active complex at saturating
substrate that will lead to the same proportional decrease in
Catalyst Structure, Function, and Design. Effective ca-
talysis, such as that provided by enzymes, will occur at active
sites which show a high degree of complementarity to the
transition state that results in strong stabilization through
electrostatic, hydrogen-bonding, and hydrophobic interactions.
The relatively rigid catalyst Zn2(1)(H2O) may interact with
either the phosphate or the uracil fragments of substrate.42-44
The interactions with the phosphate fragment will be expressed
at the ground state for binding of the diester monoanion and
strengthen on proceeding to the transition-state oxyphosphorane
dianion. The interactions between Zn2(1)(H2O) and the N3 of
the uracil group will also be expressed at the ground-state
complex. However, these interactions may be nonproductive
(Scheme 5) if they are weakened by a reorientation of the
ground-state complex on proceeding to a reaction transition state
that gives optimal electrostatic interactions between the cationic
core of the catalyst and the oxyphosphorane dianion.
the apparent values for Kd and kc, respectively, for the catalyzed
46
reaction but no change in kc/Kd ) (kZn)app
.
We suggest that the substrates UpPNP and UpU bind to the
catalyst in ground-state conformations where the interactions
with the uracil ring are relatively strong and in different
transition-state conformations that optimize the very strong
electrostatic interactions with the oxyphosphorane dianion at
the expense of weaker interactions with the uracil ring(s) (see
below). This may be a general result for small molecule catalysts
that show two or more types of stabilizing interactions with
bound substrate/transition state but lack a binding pocket that
shows a high lock-and-key complementarity to the transition
state.
(3) Electrostatic Interactions. The following observations
on the specificity of Zn2(1)(H2O) for cleavage of phosphate
diesters can be rationalized by a transition state that is stabilized
primarily by electrostatic interactions with the catalyst.
(1) There is a (4.4-7.2) kcal/mol increase in the stabilizing
interactions between Zn2(1)(H2O) and phosphate diester ligand
on proceeding from the reaction ground state to the transition
state (Figure 5). This can be rationalized by an increase in
electrostatic interactions between the cationic catalyst and
There are several advantages of modular small molecule and
enzyme catalysts with domain(s), connected by a flexible linker-
(s), that interact with different substrate fragments, over catalysts
with a rigid structure. (1) It is simpler to design such a flexible
catalyst that explores many conformations to find the one
providing maximum transition-state stabilization than a catalyst
of a single fixed structure that provides the same enthalpic
transition-state stabilization. (2) It will be difficult to change
the substrate specificity for a rigid catalyst because changes in
structure that modify the binding specificity at one domain may
also adversely affect the binding interaction at other domains.
The interdependence of interactions at such domains will be
reduced for a modular catalyst where they are free to move
independently, so as to maximize the binding interactions at
each. This will favor rapid evolution of new enzyme activi-
(46) Fersht, A. R. Structure and Mechanism in Protein Science; W. H. Freeman
and Co.: New York, 1999; pp 114-118.
9
1620 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Zn(II) Catalyst for Cleavage of RNA Analogues
A R T I C L E S
ties.47,48 (3) Conformational flexibility that reversibly exposes
the active site to bulk solvent is required for the large number
of enzymes that encapsulate their bound substrate(s).49
Effective catalysis of phosphate diester cleavage may be
obtained by tethering a cationic core to a nucleoside recognition
element, provided the tether allows sufficient conformational
flexibility for both groups to show optimal binding interactions
with their targeted regions of the substrate. A simple example
of this strategy that is being pursued in a number of laboratories
is the tethering of a metal ion complex to a short sequence of
antisense RNA that is complementary to a strand of messenger
RNA that has been targeted for cleavage.50-54
Acknowledgment. J.R.M. and J.P.R. thank the National
Science Foundation (CHE9986332) for support of this work.
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