Electrophilic assistance to the cleavage of an RNA model phopshodiester via specific and general base-catalyzed mechanisms

Article abstract of DOI:10.1021/jo301649u

Kinetics of transesterification of the RNA model substrate 2-hydroxypropyl 4-nitrophenyl phosphate promoted by Mg²⁺ and Ca²⁺, the most common biological metals acting as cofactors for nuclease enzymes and ribozymes, as well as by Co(

Full text of DOI:10.1021/jo301649u

Article
pubs.acs.org/joc
Electrophilic Assistance to the Cleavage of an RNA Model
Phopshodiester via Specific and General Base-Catalyzed Mechanisms
†
‡
,‡
David Octavio Corona-Martínez, Paola Gomez-Tagle, and Anatoly K. Yatsimirsky*
†
Universidad de Sonora, Campus Cajeme, Division
́
de Ciencias Biolog
́
icas y de la Salud, 85000 Ciudad Obregon
́ ́
, Sonora, Mexico
‡
Facultad de Química, Universidad Nacional Auton
́
oma de Mexico, 04510 Mex
́
́
ico D.F., Mexico
́
*
S Supporting Information
ABSTRACT: Kinetics of transesterification of the RNA model substrate 2-
hydroxypropyl 4-nitrophenyl phosphate promoted by Mg2+ and Ca , the most
common biological metals acting as cofactors for nuclease enzymes and ribozymes, as
2+
3
+
3+
+
+
well as by Co(NH3)6 , Co(en)3 , Li , and Na cations, often employed as mechanistic
probes, was studied in 80% v/v (50 mol %) aqueous DMSO, a medium that allows one
−
to discriminate easily specific base (OH -catalyzed) and general base (buffer-catalyzed)
2+
+
reaction paths. All cations assist the specific base reaction, but only Mg and Na assist
2
+
the general base reaction. For Mg -assisted reactions, the solvent deuterium isotope
effects are 1.23 and 0.25 for general base and specific base mechanisms, respectively.
2
+
Rate constants for Mg -assisted general base reactions measured with different bases fit
the Brønsted correlation with a slope of 0.38, significantly lower than the slope for the
unassisted general base reaction (0.77). Transition state binding constants for catalysts
⧧
in the specific base reaction (K OH) both in aqueous DMSO and pure water correlate
with their binding constants to 4-nitrophenyl phosphate dianion (K ) used as a minimalist transition state model. It was found
NPP
⧧
3+
3+
3
⧧
2+
2+
that K
≈ K
for “protic” catalysts (Co(NH3)6 , Co(en) , guanidinium), but K
Lewis acids. It appears from results of this study that Mg is unique in its ability to assist efficiently the general base-catalyzed
transesterification often occurring in active sites of nuclease enzymes and ribozymes.
≫ K
for Mg and Ca acting as
OH
NPP
OH
NPP
2+
INTRODUCTION
Scheme 1
■
Rapidly growing information on detailed mechanisms of action
of nuclease enzymes and ribozymes reveals a large diversity of
possible roles played by metal ions and acid−base functional
1
groups in the transition state stabilization. An essential part of
all proposed mechanisms is the electrophilic assistance to the
nucleophilic attack on the phosphoryl group provided either by
2
+
2+
2+
a Lewis acid (typically Mg , Ca , or Zn ) or by a protic acid
imidazolium, ammonium, or guanidinium groups). The latter
can operate as a general acid catalyst by proton transfer or
provide the electrostatic stabilization for the anionic transition
state.
(
only at very high concentrations of bases, which significantly
affect the reaction medium and make rather ambiguous the
1c,5
interpretation of kinetic results.
In particular, enzyme-like
mechanisms involving electrophilic assistance to general base
mechanism of transesterification by a protonated base or by a
metal ion reported in earlier studies were not confirmed later
In most cases the RNA cleavage occurs by the action of 2′-
OH ribose functionality as an internal nucleophile. In biological
systems the nucleophilic attack is assisted by general base
catalysis, but chemical transesterification/hydrolysis of phos-
phodiesters possessing a β-hydroxyl group proceeds mostly
through equilibrium predissociation of hydroxyl group with
subsequent nucleophilic attack of the alkoxide on the
6
and appeared to be artifacts of medium effects or metal
7
complexation by a base as a ligand, respectively. Recently, it
has been shown that changing the reaction medium from water
to 80% v/v (50 mol %) aqueous DMSO leads to a strong
suppression of the specific base hydrolysis at a given pH,
allowing direct observation of the general base reaction path at
much lower base concentrations and free of possible
2
,3
phosphodiester group (Scheme 1), which can be formally
considered as a specific base mechanism. It has been shown that
metal complex catalysis in such reactions involves an electro-
philic assistance to the last step by the aquo form of the
complex (M in Scheme 1), whereas the hydroxo form of the
8
complications. Under these conditions it was possible to
observe for the first time a clear “bell-shaped” second-order in
catalyst kinetics of buffer catalysis reminiscent of the classical
4
complex is inactive.
A slow general base-catalyzed path does exist but can be
Received: August 3, 2012
detected in the background of much faster specific base reaction
Published: September 19, 2012
©
2012 American Chemical Society
9110
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Scheme 2
5
−1
8
mechanism of RNA-se A. In this paper we apply a similar
approach to analyze the Lewis acid assistance to the general
base catalysis by some of the most common cations acting as
and kB = 1.4 × 10− M−1 s . Therefore the maximum
contribution of the specific base catalysis to the observed rate
constant of transesterification in the absence of metal ion is
2+
−
−9 −1
cofactors for nuclease enzymes and ribozymes, namely, Mg
kOH[OH ] = 1.9 × 10 s . The corresponding contribution of
2+
+
+
−6 −1
and Ca . Also the effect of alkali cations (Na , Li ) was studied
for comparison.
the general base catalysis is k [B] = 1 × 10 s , and obviously
B
this is the principal reaction path in the absence of metal ions.
However, the dianionic transition state of the specific base
reaction can be stabilized much more efficiently than the
monoanionic transition state of the general base reaction by the
electrophilic assistance, and this can make the contribution of
the specific base reaction significant in the presence of metal
ions or other electrophilic catalysts.
A particular case of electrophilic assistance is the outer-
sphere mechanism proposed for some Mg(II)-dependent
2
+
enzymes and ribozymes where the aqua-ion Mg(H O)6
2
provides the transition state stabilization by hydrogen bonding
1b,d,9
to protons of coordinated water molecules.
Chemical
catalysis in phosphodiester cleavage by protic acids is generally
much less efficient than that achieved by Lewis acids, and for
this reason it seems surprising that a kinetically labile metal ion
may act via an outer-sphere rather than an inner-sphere
mechanism. The most common experimental test for the outer-
sphere mechanism is the retention of catalytic activity when
Mg(II) is substituted with a kinetically inert complex such as
A general expression for the observed first-order rate
constant of HPNP transesterification has the form
−
kobs = kOH[OH ] + k [B] + kB,BH[B][BH]
B
−
+
kM,OH[M][OH ] + kM,B[M][B]
(1)
3+
9,10
Co(NH3)
6
.
Surprisingly, the efficiency of catalysis by
The second-order rate constant of the catalytic reaction k was
calculated as the slope of the plot of kobs versus catalyst
concentration [M] at fixed pH, eq 2:
2
Co(III) ammine complexes was never compared with that of
Mg(II) in model systems, and for this reason we included in
3+
this study also an investigation of catalysis by Co(NH ) and
3
6
Co(en)33 complexes.
+
dkobs
−
k2 =
= kM,OH[OH ] + kM,B[B]
A simple RNA-type model substrate 2-hydroxypropyl 4-
nitophenyl phosphate (HPNP) was employed, which allows
one a convenient spectrophotometric monitoring of the rate of
intramolecular transesterification. Low basicity of the 4-
nitrophenolate makes unlikely any kind of electrophilic leaving
group assistance either by a protic or a Lewis acid. Therefore
the most probable mechanism of catalysis with HPNP as a
substrate is binding of metal ion or a proton donor to
equatorial oxygens in the phosphorane transition state as
illustrated in Scheme 2 for cooperation of the metal ion with
specific base (1) or general base (2) reactions (encircled
negative charges refer to phosphate parts of the transition states
and are not the total charges).
Studies in a mixed aqueous DMSO solvent were
complemented with studies in pure water where only the
specific base reaction was observed. In conclusion we analyze
correlations between transition state stabilization free energy
and binding free energy of the catalysts to a ROPO32− dianion
considered as a crude transition state model for the specific
base reaction (3 in Scheme 2).
d[M]
(2)
The individual rate constants kM,OH and kM,B that correspond to
cooperation of the metal ion or other electrophilic catalyst with
specific base or general base catalysis, respectively, were
determined from two series of experiments. In the first series,
rate constants k were measured in buffer solutions of constant
2
total buffer concentration, but variable proportions of
protonated (BH) and neutral (B) forms. In this case both
the concentration of free base and the concentration of free
hydroxide anions are variable since they are related by eq 3:
⎛
⎞⎛
⎞
⎟
Kw
[B]
K ⎠⎝ [BH] ⎠
−
[OH ] = ⎜ ⎟⎜
⎝
(3)
a
If only one term in eq 2 is significant, the k must be
proportional to either [B] or [B]/[BH] with coefficients of
proportionality kM,B or kM,OH, respectively. If both terms are
significant the k must be a nonlinear function of either [B] or
B]/[BH], and the respective rate constants can be calculated
2
2
[
from eq 2 by multiparameter correlation. In the second series
rate constants k were measured in a buffer solution of constant
2
RESULTS AND DISCUSSION
■
[B]/[BH] ratio but variable total buffer concentration. In this
case, only the concentration of free base is variable, and the
concentration of free hydroxide anions is constant and is
determined by eq 3. Equation 2 now allows one to calculate
Catalysis in 80% v/v Aqueous DMSO. The majority of
kinetic measurements were performed in 0.1 M piperidine/HCl
buffer solutions containing from 10% to 70% free base. This
corresponds to variation in pH from 8.9 to 10.3. With pK of
kM,B from the slope of the dependence of k on total buffer
w
2
1
8.77 in this medium these are essentially neutral solutions with
concentration and k
from the intercept.
M,OH
−10
2+
very low concentration of free hydroxide ions from 1.2 × 10
Results for Ca in 0.1 M piperidine buffer with various
degree of neutralization from 10% to 70% free base are shown
in Figure 1. As one can see from Figure 1a, the reaction in the
to 3.2 × 10− M. The rate constants of specific base and general
9
base-catalyzed reactions in this medium are kOH = 0.58 M−
1
s
−1
9
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Article
Figure 1. Catalysis by Ca2+ in 0.1 M piperidine buffer in 80% v/v DMSO. (a) Plots of kobs versus metal ion concentration. (b) Plots of k versus
2
concentration of free base [B] (solid squares, lower scale) or versus [B]/[BH] (open squares, upper scale).
Table 1. Kinetic and Thermodynamic Parameters of HPNP Transesterification in 80% v/v DMSO at 37°C (B = Piperidine)
2
kM,OH (M−2 s−1
)
log K⧧
log K⧧
b
catalyst
Ca2+
Mg2+
Li+
Na+
kM,B (M− s−1
)
log KNPP
B
OH
(8.13 ± 0.3) × 106
(5.0 ± 0.5) × 107
(2.30 ± 0.06) × 104
7.14
4.5
4.63
7.9
4.6
2.5
5.9
6.2
0.46 ± 0.05
5.22
a
2.9
(1.7 ± 0.2) × 10−4
(1.70 ± 0.09) × 10
2
1.1
2.0
2.2
a
3+
(5 ± 1) × 105
Co(NH )
5.45
3
6
+
Co(En) 3
(8.4 ± 0.5) × 105
3
HGu+
0.0017
8
2.70
a
b
In 85% DMSO, from ref 11. Relative error in the range from ±0.05 to ±0.08.
Figure 2. Catalysis by Mg2+ in 0.1 M piperidine buffer in 80% v/v DMSO. (a) Plots of kobs versus metal ion concentration. (b) Plots of k versus
2
concentration of free base [B] (solid squares, lower scale) or versus [B]/[BH] (open squares, upper scale).
presence of Ca(II) is first-order in metal ion, and the
acceleration effect over the background reaction is about 103
at 0.04 M Ca(II). The second-order catalytic rate constant is
not proportional to the base concentration; however, it is
Ca(II) was confirmed by independence of the reaction rate on
total buffer concentration at 50% free base fraction in the
presence of Ca(II). Thus the electrophilic assistance by Ca(II)
leads to the actual acceleration effect of the specific base
−
6
strictly proportional to the [B]/[BH] ratio, that is, to [OH ]
reaction by a factor of 10 , but there is no detectable effect on
(
Figure 1b, solid and open squares, respectively). The third-
the general base reaction, which in the absence of metal is a
order rate constant kM,OH calculated from the slope of the line
in Figure 1b is given in Table 1. The absence of any
contribution of the general base-catalyzed reaction assisted by
1000-fold faster than the specific base reaction.
Similar results with Mg(II) are shown in Figure 2.
Measurements were performed only up to 50% free base
9
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3+
+
+
because of precipitation of Mg(OH) in more basic solutions.
Co(NH ) 3+ ≈ Co(en)3 > Li ≫ Na , which closely follows
2
3 6
The overall catalytic activity of Mg(II) is about 5 times larger
than that of Ca(II) at the same free base fraction. The reaction
is also first-order in metal ion (Figure 2a), but the plot of k2
versus base concentration has a smaller upward curvature, and
the order of decreasing charge density (Z/r ) of cations and
i
points principally to electrostatic catalysis. Only Mg and Na+
2
+
2
+
assist the general base reaction. The fact that only Mg but not
Ca can assist the general base reaction is rather surprising. Of
2+
2+
the plot of k versus [B]/[BH] ratio is curved downward
course, Mg is a stronger electrophile, but by itself that does
not mean that it should be relatively more efficient in
promoting the general base reaction. Indeed, on going from
2
(
Figure 2b). This means that both terms in eq 2 are significant.
Figure 3 shows the dependences of k on total buffer
2
+
concentration at two degrees of neutralization, which confirms
Mg2+ to the much weaker electrophile Na the rate constant
5
kM,OH decreases by a factor of 3 × 10 , but k only by a factor
of 3 × 10 , which means that for reaction assisted by Na the
general base path contributes actually more than for Mg -
assisted reaction. Among alkali metal ions, the more electro-
philic Li catalyzes only specific base reaction, but Na shows
catalytic activity in both reaction paths. Apparently, there are
some requirements most probably related to details of the
structure of the coordination sphere and hydration of metal ion
that cannot be identified at the moment, needed for efficient
electrophilic assistance in general base reaction. Interestingly,
M,B
3
+
2
+
+
+
+
guanidinium cation with a smaller charge density than Na is a
more efficient catalyst for the general base reaction, which may
be related to its high efficiency of bidentate binding to
12
phosphate anions.
A useful way to analyze catalytic effects is to see them in
terms of transition state stabilization of the unassisted
1
3,14
reaction.
The respective transition state “binding constants”
B
⧧
⧧
K
and K , which reflect pseudoequilibrium binding of an
OH
electrophilic catalyst M to the transition state of specific base or
general base reaction with formation of transition states 1 or 2
Figure 3. Catalysis by MgCl in piperidine buffer at variable total
buffer concentration in 80% v/v DMSO. Circles show results in
2
(
Scheme 2), are given by eqs 4 and 5, respectively, and
logarithms of these constants are shown in Table 1.
DMSO/H O, and squares show results in DMSO/D O.
2
2
kM,OH
⧧
K
K
=
OH
kOH
(4)
(5)
this conclusion: the slopes are nonzero, and both slopes and
intercepts of the lines increase on increasing the fraction of free
base. The average values of kM,OH and kM,B calculated from both
types of experiments are given in Table 1.
kM,B
⧧
=
B
kB
+
+
Among alkali metal ions only Li and Na showed noticeable
+
catalytic effects. In case of Li only assistance to specific base
These “equilibrium constants” multiplied by the concentration
of M give the real acceleration effect of the catalyst at this
reaction was observed (see Figure 1S in Supporting
+
Information). Catalysis by Na was very weak, but in contrast
particular concentration and therefore serve as a direct measure
⧧
to other cations sodium did not precipitate in basic solutions,
and this allowed us to measure its activity in unbuffered
solutions in the presence of a relatively high known
concentration of free hydroxide (Figure 2S in Supporting
of the efficiency of catalysis. One can notice that log K
OH
values, which refer to the formation of transition state 1 from a
dianionic transition state of specific base reaction, are
approximately twice as large as log K , which refer to the
⧧
B
Information) from which k
can be directly calculated.
formation of transition state 2 from a monoanionic transition
state of general base reaction. This is in agreement with mostly
electrostatic stabilization by the electrophilic assistance. The
absolute values of transition state binding constants for the
specific base reaction are very large. To see how large may be
the affinity of electrophilic species employed as catalysts toward
a phosphate dianion, we measured the ground state association
constants (KNPP) of some of these species with 4-nitrophenyl
phosphate (NPP) dianion giving complexes 3 (Scheme 2, R =
M,OH
Then, catalysis by Na+ was measured in piperidine buffer
Figure 3S in Supporting Information), and after correction for
(
the contribution of specific base reaction the kM,B was
calculated. Both rate constants are given in Table 1.
3
+
Measurements with Co(NH3)6 were limited by unexpect-
edly low solubility of the hydroxide, which precipitated already
at pH of about 9.5. Reaction rates measured in piperidine buffer
with less than 50% free base were independent of total buffer
concentration, and experiments at variable [B]/[BH] ratio
indicated the catalytic effect on the specific base reaction only.
Similar results were obtained with Co(en)3 (Figure 4S in
Supporting Information). The respective third-order rate
constants are given in Table 1 together with the previously
determined catalytic rate constant for guanidinium cation.
Inspection of results collected in Table 1 indicates the
following general trends. The catalytic activity in the specific
2‑
4-nitrophenyl). Dianions of phosphate monoesters ROPO3
are often considered as crude analogues of transition states of
3
+
15
phosphodiester hydrolysis/transesterification reactions. The
respective values of log KNPP are collected in the last column of
⧧
Table 1. First, one can notice that K is smaller than KNPP both
B
for M = Mg2+ and M = guanidium cation in accordance with
the larger negative charge (2−) of NPP compared to that of the
general base transition state (1−), again in line with mostly
electrostatic stabilization. Second, for the specific base reaction
2
+
2+
base reaction decreases in the order Mg > Ca
>
9
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Table 2. Solvent Deuterium Kinetic Isotope Effects in Different Reaction Paths
8
0% v/v DMSO
kOH/kOD
none
kM,OH/kM,OD
kM,BH/kM,BD
Mg2+
kBH/kBD
M =
Mg2+
Ca2+
none
a
0
.87 ± 0.02
0.25 ± 0.05
0.36 ± 0.05
1.23 ± 0.06
1.4
water
kOH/kOD
none
kM,OH/kM,OD
M =
Mg2+
Ca2+
Co(NH3)63+
0.65 ± 0.07
Guanidine H+
0
.80 ± 0.04
0.42 ± 0.06
0.47 ± 0.05
0.79 ± 0.07
a
Reference 8.
3
+
⧧
The results for Mg2+ in piperidine buffer solution containing
with Co(NH )
K
is only 3 times larger than KNPP, but
OH
3
6
with Ca2 and Mg K⧧ surpasses KNPP by more than 2
+
2+
40% free base in 80% v/v DMSO/D O are shown in Figure 3
OH
2
orders of magnitude. In other words, transition state affinity of
(solid squares). The slope of this line is smaller than that in
3+
Co(NH )
corresponds mostly to simple ion-pairing, but
H O at the same fraction of free base indicating a normal
3
6
2
2+
2+
Ca and Mg have much higher transition state affinity. A
similar, although less pronounced, effect is observed in water
although small SKIE in the general base reaction, but the
intercept is larger pointing to an inverse isotope effect in
−
(
see below).
specific base reaction. In fact, the concentration of free OD is
To get further insight into mechanisms of different reaction
2
.8 times smaller in the mixture with D O than the
2
−
paths, solvent kinetic deuterium isotope effects (SKIE) on
respective rate constants were measured, Table 2.
concentration of free OH in the mixture with H O at the
2
same fraction of free base because of a larger isotope effect in
pK than in pK of piperidinium, and the real isotope effect in
The SKIE for specific base reaction in the absence of metal
ions (kOH/kOD) was determined by measurements in
w
a
kM,OH is significantly larger than it seems from the raw results in
the Figure 3. The calculated isotope effects for both reactions
−
unbuffered solutions in the presence of 1−3 mM OH or
−
OD in DMSO mixtures with H O or D O, respectively. The
2+
2
2
are given in Table 2. The isotope effect for Ca -catalyzed
observation of an inverse SKIE for this path agrees with
reaction was determined in a similar way and also is shown in
reported for cis-4-hydroxytetrahydrofuran 3-phenyl phosphate
Table 2.
3
kOH/kOD = 0.51 in water at 50 °C and kOH/kOD = 0.54 for
The SKIE in general base reaction assisted by Mg2+ is
1
6,17
alkaline cleavage of HPNP at 25 °C.
This effect has several
somewhat smaller than that for unassisted reaction with
piperidine base (Table 2). Rather small by itself, SKIE for
unassisted reaction is consistent with an early transition state
expected for a reaction with strongly activated leaving group,
and a smaller value in the presence of Mg2+ points to an even
earlier transition state for the catalytic reaction in agreement
with decreased Brønsted slope for the latter (see below).
Opposite SKIEs for kM,OH and kM,B rate constants confirm the
correct assignments of the reaction paths based on formal
kinetic study in the buffered systems. Isotope effects in specific
base reaction are discussed later together with results in water.
Relatively high catalytic activity of Mg2+ in the general base
reaction allowed us to determine kM,B with this cation for
contributions. In accordance with mechanism in Scheme 1
(
with [M] = 0) the expression for kOH is given by eq 6, and the
expression for the isotope effect takes the form of eq 7.
k0KaSH
kOH
=
Kw
(6)
(7)
H
SH
SD
D
H
⎛
⎜
⎞⎛
⎟⎜
⎞⎛
⎟⎜
⎞
⎟
⎠
kOH
kOD
k0
Ka
Kw
=
D
⎝ k ⎠⎝ Ka ⎠⎝ K
0
w
The isotope effects in the acid dissociation constant of the
substrate (hydroxyl group of HPNP) and in the autoprotolysis
different bases. Figure 4 shows the dependences of k for
obs
constant of water (K ) likely compensate for each other, and
w
2+
H
0
D
0
HPNP transesterification in the presence of 6 mM Mg on free
the observed isotope effect should be close to k /k . The
value k /k = 0.95 was reported for transphosphorylation of
H
D
base concentration obtained from experiments with variable
total concentration of different buffers: 3-methoxypropylamine
(3-MeOPA), 2-methoxyethylamine (2-MeOEA), morpholine,
p-dimethylaminopyridine (DAP), and imidazole. All buffers
except imidazole were employed at constant 30−70% fraction
of the free base. Imidazole, which has very low basicity in this
medium (see Table 2), was added as a free base to 0.1 M 3-
methoxopropylamine buffer containing 30% free base (pH
0
0
5
′-UpG-3′ measured at pH 14 when the substrate hydroxyl is
18
completely deprotonated.
2
+
Isotope effects in the Mg -catalyzed reaction were measured
in piperidine/HCl buffer solution in order to separate effects in
D
specific base and general base reactions. Values of pK = 10.83
a
D
±
0.03 for piperidinium cation and pKw = 20.25 ± 0.05 for
autoprotolysis of water were obtained by potentiometric
titrations of piperidinium chloride and DCl, respectively, in
1
0.0). The pKa values for all buffers determined by
potentiometric titrations of their hydrochlorides in 80% v/v
DMSO at 37 °C are given in Table 3 together with respective
8
0% v/v DMSO/D O at 37 °C with 0.1 M Me NCl supporting
2
4
electrolyte. Previously reported respective values in 80% v/v
DMSO/H O are 9.88 and 18.77. Thus, the isotope effects in
8
k
M,B values.
2
the acid dissociation constants in the mixed solvent are
approximately 0.5 logarithmic units larger than those typically
Figure 5 shows the results for kM,B (solid squares), which fit
to eq 8. For comparison also are shown previously reported
plots for guanidinium-assisted reaction (open squares, slope =
0.69) and unassisted general base reaction (solid circles, slope =
0.77).
19
observed in pure water. With these values one can calculate
−
the concentration of free OD in the buffer at a given fraction
of free base.
9
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Article
in neutral solutions with reactivity typical for much stronger
bases, which in neutral solutions are mostly protonated.
Catalysis in Water. The catalytic activity of alkaline earth
cations in water is very low and can be conveniently measured
at high free hydroxide concentrations. For this reason the
2+
3+
3
3+
catalytic rate constants for Ca , Co(en) , and Co(NH3)6
were determined in unbuffered solutions at variable concen-
2
+
tration of Me NOH in the range 1−10 mM. Since Mg
4
precipitates at pH above 10, its reactivity was measured in
.1 M Tris buffer at pH 9−9.5. The reactivity of guanidinium
0
chloride was measured in 0.1 M piperidine buffer in a pH range
of 10.5−11.5 to avoid uncertainty in free hydroxide
concentration, which may be reduced by partial deprotonation
of guanidinium cation in highly basic solutions. The general
base catalysis in HPNP transesterification in water is
undetectable in 0.1 M and less concentrated buffer solutions
in the background of much faster specific base reaction. In all
cases we observed a simple first-order in catalyst and first-order
in hydroxide kinetics (Figure 5S in Supporting Information)
with third-order catalytic rate constants kM,OH shown in Table
4. Solvent deuterium isotope effects shown in Table 2 were
determined for these rate constants under similar conditions.
Figure 4. Observed rate constants in the presence of 6 mM Mg2+
versus concentrations of different bases. [B] = [free base].
Table 3. pK Values of Protonated Forms of Different Bases
a
and the Third-Order Rate Constants for HPNP
2
+
Transesterification in the Presence of Mg in 80% v/v
DMSO
kM,B (M− s−1
2
)
pKa
Table 4. Third-Order Rate Constants for HPNP
piperidine
9.88
9.65
9.1
0.46 ± 0.05
0.36 ± 0.03
0.15 ± 0.02
0.053 ± 0.003
0.064 ± 0.013
0.008 ± 0.004
Transesterificaiton in Water and Stability Constants of 4-
3
2
-MeOPA
-MeOEA
Nitrophenyl Phosphate Complexes (KNPP
)
2
log K⧧
catalyst
HGu+
Co(NH )
kM,OH (M− s−1
)
log KNPP
OH
morpholine
DAP
8.03
a
0.37 ± 0.03
26.7 ± 0.5
61.7 ± 0.6
85.2 ± 2.4
153 ± 7
0.36
2.31
2.68
2.82
3.07
4.71
0.54
7.46 ± 0.03
5.4
3+
6
1.97 ± 0.04
2.09 ± 0.06
1.51 ± 0.03
1.53 ± 0.03
3
imidazole
Co(En)33+
Ca2+
Mg2+
Zn2+
b
6.7 × 103
1.76
c
a
b
c
Reference 8. Reference 20, at 25 °C. Reference 21, at 25 °C.
There is an uncertainty in the case of guanidinium because
the cation-assisted specific base mechanism is kinetically
indistinguishable with general base catalysis by the neutral
guanidine molecule. The interrelation between the rate
constants is given by the eq 9 where B and BH are neutral
BH
and protonated guanidine and Ka is the acid dissociation
constant of guanidinium cation.
⎛
⎜
⎝
⎞
⎟
⎠
Kw
kB = kBH,OH
BH
Ka
(9)
22
With pK 13.3 at 37 °C for guanidinium cation one obtains kB
a
−1
−1
−1
=
s
0.19 M
s
in water, which is not far from k = 0.025 M
for guanidine free base in 80% v/v DMSO reported in ref 8.
B
2+
−1
Figure 5. Brønsted plots for Mg -assisted general base-catalyzed
transesterification of HPNP in 80% DMSO. Data for guanidinium-
assisted and unassisted reactions are from ref 8.
However, the latter rate constant has the normal solvent
isotope effect of 1.7, while the reaction in water has the inverse
8
isotope effect (Table 2) typical for specific base reaction.
The inverse SKIE measured for kOH is smaller than the
log kM,B = −4.1 ± 0.3 + (0.38 ± 0.03)pKa
(8)
16,17
reported value 0.54 at 25 °C for the same HPNP substrate.
Repeating measurements at 25 °C we found a closer value of
.74, which however is still significantly different from the
The slope of the Brønsted plots progressively decreases on
going from unassisted general base reaction to guanidinium-
and magnesium(II)-assisted reactions in accordance with
expected shift to earlier transition state for stronger electro-
philically activated reactions. An important consequence of this
trend is that even weak bases such as imidazole and probably
even nucleobases may participate in catalysis assisted by Mg2+
0
reported one. A probable reason is different methodology of
measurement. The value reported in ref 16 was determined
from rate constants measured in H O and D O at pH = pD
2
2
with pD calculated by the usually applied correction factor 0.4,
while we calculated the isotope effect as the ratio of second-
−
order rate constants measured by varying total [OH ] or total
9
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−
H
D
D
H
[
OD ] taken at high excess over [HPNP] in respective
⎛
⎞
⎟
⎠
⎛
⎜
⎞
⎟
⎠
kM,OH
k2
Kw
solvents. There are concerns that the correction term for pD
actually depends on temperature and glass properties of the
electrode and is not a universal constant.
with known total concentration of hydroxide are free from this
possible error. In any case, it is clear that the general base
reaction has a significant inverse SKIE.
Other relevant reported data involve SKIE 1.43 for HPNP
cleavage in the presence of a Zn(II) complex at high pH when
= ⎜
kM,OD
⎝ k2
⎝ K
w
pH=pD
(16)
3,23
Measurements
D
w
H
w
24
H
D
2
With K /K = 0.135 at 25 °C one obtains from k /k
3.2 the value of kM,OH/kM,OD = 0.43 for Zn(II)-catalyzed
cleavage of UpPNP, which is very close to SKIE determined for
Mg - and Ca -catalyzed HPNP cleavage (Table 2).
Considering results shown in Table 2 for the specific base
reaction both in aqueous DMSO and water together with the
above cited literature data one concludes that the inverse SKIE
in general becomes stronger in the presence of metal ions
providing the Lewis acid assistance. Reactions in the presence
of “protic” electrophilic catalysts Co(NH3)6 and guanidinium
cation have only a little bit larger inverse isotope effects than
the unassisted reaction. The inverse solvent deuterium effect is
=
2
2+
2+
16
the complex is totally converted into the hydroxo form and
SKIE reported for transesterification of the chemically similar
to HPNP substrate uridine 3′,4-nitrophenyl phosphate
(
UpPNP) catalyzed by a Zn(II) binuclear complex at variable
4
3+
pH. For the latter system SKIE is 0.8 at high pH when the
complex is mostly in hydroxo form and 3.2 at pH = pD = 7.1
when the complex is mostly in the aquo form. Assuming that in
both systems the reaction proceeds in accordance with the
mechanism in Scheme 1 and the active form of the catalyst is
the aquo complex, which undergoes deprotonation to inactive
hydroxo complex (eq 10) with dissociation constant Ka,M, the
expression for the observed second-order rate constant of the
catalytic reaction takes the form of eq 11.
25
normally observed in specific base reactions and is attributed
to the fact that hydroxide anion is a stronger base in D O than
in H O. This should be valid also for the alkoxide anion. An
2
26
2
increase in the isotope effect in the presence of metal ions may
be related to additional inverse equillibrium isotope effect of
metal ion association with the anionic transition state. To test
this hypothesis we measured the association constants of Mg
with NPP dianion in H O and D O under similar conditions
(37 °C, ionic strength 0.1M) and obtained log KNPP = 1.53 ±
0.01 and log KNPPD = 1.63 ± 0.01, which correspond to a
modest inverse solvent isotope effect of 0.79 ± 0.03. Stronger
inverse equilibrium isotope effects are observed, however, for
hydroxide association with metal ions. Indeed, the deprotona-
tion equilibrium (9) is equivalent to the equilibrium of
association of the metal ion (or metal complex) with hydroxide
2
+
n+
⇄ M(OH)n−1 + H+
M
(10)
2
2
H
SH
⎛
Ka
⎞
+
⎜
[H ] ⎟
k2 = k
C⎜ 1 + K
⎟
a,M
⎜
⎟
+
⎝
[H ] ⎠
(11)
Under conditions of complete deprotonation of the catalyst
at high pH eq 10 takes the form of eq 12, and the expression for
SKIE is eq 13.
anion with the equilibrium constant KMOH = Ka,M/K . The
w
equilibrium isotope effect in K
typically about 3, divided by the isotope effect 7.4 in K ,
equals therefore that in Ka,M
,
⎛
⎝
SH ⎞
a,M ⎠
MOH
Ka
K
4
24
k = k ⎜
⎟
⎟
w
2
C⎜
which gives the inverse isotope effect about 0.4. A similar effect
may be expected for association between metal ions and
alkoxide anions and therefore we consider the increased inverse
SKIE in specific base reaction assisted by metal ions as an
evidence of the contact between the metal ion and entering
alkoxide nucleophile in the transition state. This hypothesis
finds some support from the correlation analysis between K
and KNPP values discussed below.
Correlation between Transition State “Binding Con-
stants” and Binding Constants of Catalysts to a
Transition State Analogue. Table 4 contains the stability
constants of NPP complexes measured under similar conditions
(12)
H
H
⎞⎛
SH
SD
⎞⎛
D
H
⎞
⎠
⎛
k2
kC
Ka
K
a,M
a,M
=
⎜
⎟⎜
⎟⎜
⎜
⎟
⎟
k2D
C
D
⎝ k ⎠⎝ Ka ⎠⎝ K
(13)
In eq 13 equilibrium isotope effects in dissociation constants of
the substrate and catalyst partially compensate for each other,
but since the substrate is a much weaker acid its dissociation
⧧
OH
19
constant is expected to have a larger isotope effect and
therefore the actual SKIE in catalytic rate constants kCH/kCD
should be a smaller number than the observed SKIE. This
means that for Zn(II)-catalyzed cleavage of UpPNP kCH/k is
D
C
2+
smaller than 0.8, and for catalytic HPNP cleavage kCH/kC
most probably is below unity.
D
and includes also literature data for Zn for comparison.
Inspection of the results in Table 4 shows that the catalytic
activity of Co(III) complexes operating through the outer-
sphere mechanism is 2 orders of magnitude larger than that of
guanidinium cation, and accordingly these complexes have
much higher affinity to the transition state model NPP than the
guanidinium cation. However, Co(III) complexes also bind
Under conditions of negligible deprotonation of the catalyst
at low pH eq 11 takes the form of eq 14:
SH
SH
⎛
⎞
⎛
⎞
Ka
Ka
−
−
k = k ⎜
⎟ = k ⎜
⎟[OH ]=k
[OH ]
2
C
+
C
M,OH
⎝
[H ] ⎠
⎝ KW ⎠
(14)
2+
2+
2+
NPP tighter than Ca , Mg , and even Zn but have much
lower catalytic activity. To see a more general picture we
attempted to correlate log K
It follows from eq 14 that
⧧
versus log KNPP for all the
OH
SH
⎛
⎞
⎟
⎠
k2
OH ]
Ka
reported so far catalytic systems for HPNP transesterification
for which the respective data are available. Besides the results
collected in Tables 1 and 4 these involve data for Zn(II)
complexes shown in Chart 1. The phosphate monoesters
employed as transitions state models for these complexes were
kM,OH = [
= k ⎜
−
C
⎝ K
W
(15)
H
D
The SKIE calculated as the ratio k /k at pH = pD equals
2
2
H
D
SH
SD
(
kC /k )(K /Ka ) and it is easy to see from the eq 15 that
C a
‑
2‑
2‑
the respective SKIE in the third-order rate constant kM,OH can
be calculated according to eq 16:
MeOPO32 or PhOPO3 instead of NPP (4-O NC H OPO );
2 6 4 3
2+
2+
2+
however, the binding constants of Mg , Ca , and Zn to
9
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Article
Chart 1
these ligands vary within limits of a factor of 2.5 for a given
metal ion, which is insignificant in the scale of the observed
catalytic effects.
Figure 6 shows the whole set of data. Red points correspond
to results obtained in 80% v/v DMSO, and other results are for
Interestingly, Ca2+ and Mg2+ in aqueous DMSO fall on this
2
1
+
2+
2+
line, while Li in DMSO and Ca and Mg in water occupy
intermediate positions. The unity slopes of both lines mean that
within each family of catalysts the improvements provided by
ligand or medium effects to the catalytic activity (transition
state binding) are of the same type, which is required for better
binding of dianion of phosphate monoester, but there is an
intrinsic difference in the type of binding between these two
groups, which makes possible the approximately 3 orders of
magnitude stronger transition state binding for the catalysts of
the second group. The most evident structural difference
between the transition state and its model is that in the former
the negative charge is distributed not only over phosphoryl
oxygens, but to a large extent is localized also on the entering
alkoxide oxygen. It is possible therefore that the advantage of
2
+
2+
Zn , coordinative unsaturated Zn(II) complexes, Mg and
2+
Ca cations is their ability to polydentate coordination, which
allows them to make use of an additional donor atom as
illustrated schematically in structure 4. This hypothesis agrees
2+
2+
with increased inverse SKIE of Mg - and Ca -assisted specific
base reactions (see above).
Figure 6. Correlation between binding constants of catalysts to the
⧧
transition state (K OH) and to a transition state model (KNPA) for
transesterification of HPNP via specific base mechanism. Red points
correspond to results in 80% v/v DMSO. Data for Zn(II) complexes
(
Chart 1) are from refs 15 and 20.
An additional observation worth mentioning is that the
transition state stabilization by Mg2+ in aqueous DMSO is
similar to that of most active Zn(II) complexes in water,
indicating the importance of solvation effects for electrophilic
assistance to nucleophilic substitution at the phosphoryl group.
Finally, the utility of using phosphate monoesters as transition
state analogues is manifested also in the observation of a good
correlation between catalytic activity and affinity of metal ions
aqueous solutions. The solid line corresponds to the situation
when log K
all “protic” electrophiles (guanidinium, Co(NH ) , Co-
(
⧧
= log KNPP. The catalysts that fit to this line are
OH
3
+
3
6
3+
+
en)31 ), Na , and Zn(II) complexes with tetradentate ligands
(
ZnL and ZnPy). All these species bind the transition state of
specific base reaction with the same affinity as the phosphate
monoester, which can be interpreted in terms of close similarity
between the binding modes in complexes 1 and 3. These
species seem to be unable to use for transition state binding
1a
to a phosphate monoester for RzB HH ribozyme cleavage.
CONCLUSION
The major point of this study is the demonstration of the
unique ability of Mg to assist the general base-catalyzed
■
more donor atoms than equatorial phosphorane oxygens. The
correlation spans over 5 orders of magnitude in K
⧧
2+
and KNPP
OH
and includes results both for water and aqueous DMSO.
transesterification of a phosphodiester. Such a mechanism is
1f
On the other hand, Zn(II) complexes with tridentate ligands
considered for, e.g., colicin E7 and I-Pol nucleases, as well as
2
3
4
2+
27
(
ZnL , ZnL , ZnL ), free Zn aquocation, and Zn(II)
for self-cleaving ribozymes. The most important aspect of this
complexes “enforced” by binuclearity (Zn L) or by incorpo-
ration of additional proton donor groups in ortho positions of
assistance effect is the low slope of the Brønsted plot, which
makes possible an efficient catalysis by weakly basic catalysts.
This may have implications for ribozyme catalysis, explaining
why such poor bases as the nucleobases can be catalytically
active. The correlation analysis based on comparison of
transition state “binding constants” and ground state binding
2
pyridine ligands (ZnPyNH ) clearly form another correlation
2
line shown by a dashed line shifted upward by 2.7 logarithmic
units. In other words, catalysts of this series bind the transition
state 500 times stronger than the monoester analogue.
9
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The Journal of Organic Chemistry
Article
constants of catalysts to a transition state analogue previously
employed for some particular catalysts proved to be a useful
tool for identification of the nature of catalyst−transition state
interaction with catalysts of different types and may find further
applications in mechanistic analysis of chemical and biological
phosphate ester cleaving systems.
hydroxy-2-propyl isomer, which is ca. 10 times more reactive than the
3
4
main 2-hydroxypropyl isomer. The presence of this minor isomer
affects significantly the initial reaction rate used for studies of slow
kinetics. For this reason we used for measurements of the initial rates
the stock solutions of HPNP prehydrolyzed by ca. 30% to reduce the
content of the rapidly reacting isomer to a negligible amount. The
integral kinetics were fitted to the eq 17 where ΔA is the observed
change in the absorbance at time t, ΔA and ΔA are the absorbance
Solvent kinetic isotope effects serve well to confirm the
assignment of general base and specific base reaction paths in
the presence of metal ions inferred from formal kinetic studies.
A small decrease by ca. 15% in SKIE for the general base
1
2
changes due to complete hydrolysis of minor and major isomers,
respectively, kfast and kslow are the first-order rate constants of
hydrolysis of these isomers, and t is the reaction time. The value of
kslow for the major slower reacting isomer was considered as the
observed first-order rate constants (kobs) for the transesterification of
HPNP.
2
+
reaction in the presence of Mg most probably reflects an
earlier transition state of the catalytic reactions, in agreement
with a decreased Brønsted slope. A significant increase in the
inversed SKIE for Lewis acid assisted specific base reaction can
be a general phenomenon in phosphodiester transestericiation,
but the origin of this effect is not entirely clear. At least partly it
can be attributed to inverse equilibrium solvent isotope effect in
metal ion−anionic transition state association by analogy with
inverse isotope effects observed for metal ion association with
phosphate monoester and hydroxide anions, but the reason for
the latter is uncertain. The major contribution to inverse SKIE
in specific base reactions comes from the anionic nucleophile
−kfastt
−kslowt
ΔA = ΔA (1 − e
) + ΔA (1 + e
)
(17)
1
2
Spectrophotometric Titrations. Determinations of association
constants of Mg2 and Ca with p-O NC H OPO is 80% v/v
+
2+
2‑
2
6
4
3
DMSO were performed by spectrophotometric titrations of 0.1 mM p-
O NC H OPO H in morpholine buffer at pH 8.0 and 37 °C by metal
2
6
4
3
2
salts. The observed association constants were corrected for the degree
8
of protonation of the phosphate monoester with known pK = 10.6
a
of p-O NC H OPO H under these conditions.
2
6
4
3
2
2
5
solvation. Probably similar solvation effects of the anionic
transition state are responsible for the above mentioned
equilibrium effect.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Rate constants for Li -, Na -, and Co(en) 3+-assisted reactions
+
+
3
in 80% v/v DMSO as function of free base fraction; rate
constants for all catalysts studied in water as function of free
hydroxide concentration. This material is available free of
charge via the Internet at http://pubs.acs.org.
EXPERIMENTAL SECTION
General Experimental Methods. 2-Hydroxypropyl 4-nitophenyl
■
phosphate (HPNP) was prepared as the barium salt according to the
2
8
literature procedure. Guanidinium chloride, piperidine hydro-
chloride, other amines employed as buffers, metal salts, Me N-
4
AUTHOR INFORMATION
Corresponding Author
*E-mail: anatoli@unam.mx.
Notes
(
OH)·5H O, Me NCl, D O (99.9% D), and p-O NC H OPO Na
■
2
4
2
2
6
4
3
2
were used as supplied. DMSO (Baker) was purified by distillation over
CaO followed by 72 h sequential drying over 4 Å molecular sieves.
2
9
Potentiometry. Potentiometric titrations were performed in a 30-
mL thermostatted cell kept under nitrogen at 37 ± 0.1 °C with 0.01 M
The authors declare no competing financial interest.
Me NCl as background electrolyte. Experimental details and
4
procedure for the electrode calibration were the same as in refs 30
and 31. The program Hyperquad 2003 was used to calculate all
32
ACKNOWLEDGMENTS
Financial support by DGAPA-UNAM (project IN 203408) is
gratefully acknowledged.
■
equilibrium constants. Determinations of association constants of p-
2
‑
3+
O NC H OPO with all cations in water and with Co(NH3)6 in
2
6
4
3
aqueous DMSO were performed by titrations of 1−10 mM p-
O NC H OPO H (obtained by passing the sodium salt through a
2
6
4
3
2
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The Journal of Organic Chemistry
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