Steric effects on the catalytic activities of zinc(ii) complexes containing [12]aneN3 ligating units in the cleavage of the RNA and DNA model phosphates

Article abstract of DOI:10.1039/c2ob25624j

A series of N-methylated mono- and di-[12]aneN₃ ligands (5-9) have been synthesized and characterized. The steric effects on the catalytic activities of their mononuclear and dinuclear zinc(ii) complexes in the cleavage of a RNA model 2-hydroxy

Full text of DOI:10.1039/c2ob25624j

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PAPER
Steric effects on the catalytic activities of zinc(II) complexes containing [12]
aneN ligating units in the cleavage of the RNA and DNA model phosphates†
3
a
a
a
a,b
c
Yang Song, Ju Zan, Hao Yan, Zhong-Lin Lu* and Ruibing Wang
Received 26th March 2012, Accepted 28th May 2012
DOI: 10.1039/c2ob25624j
A series of N-methylated mono- and di-[12]aneN ligands (5–9) have been synthesized and characterized.
3
The steric effects on the catalytic activities of their mononuclear and dinuclear zinc(II) complexes in the
cleavage of a RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP, 3) and a DNA model
methyl 4-nitrophenyl phosphates (MPNPP, 4) in methanol have been investigated at 25 °C. In the
cleavage of phosphate 3 catalyzed by the mononuclear complexes, derived from the N-methylation in the
[
12]aneN backbone, the plots of k versus [Zn(II)] changed from an upward curvature to linearity with
3 obs
increasing level of methylation, indicating that N-methylations led to a reduction of dinuclear association
that was responsible for the synergetic effect. Compared to the activities of the complex with non-
methylated di-[12]aneN ligand, those of the dinuclear zinc(II) complex (Zn -8), which has the two
3
2
N-methyl groups, were reduced by two orders of magnitude as measured by the second-order rate
constants and synergetic effect in the cleavage of both model compounds. For reactions catalyzed by the
fully N-methylated dinuclear complex (Zn -9), no synergetic effect was observed. Nevertheless, complex
2
9
–10
Zn -8 still showed the remarkable catalytic efficiency, with rate accelerations of 10 -fold in the
2
cleavage of each of the two phosphates relative to the background reactions, and the synergetic effects of
up to 561 folds. pH jump experiments confirmed that the rate-limiting step in the cleavage of 3 by Zn -8
2
involved the binding process, while that in the reaction with 4 was the chemical cleavage of the P–O
bond. Steric effects in the cleavage reactions were analyzed in detail and were compared with the
electronic effect caused by oxy anion bridging group in the di-[12]aneN ligand and also with the
3
hydrophobic effect observed in other systems. The work has further confirmed that the combination of the
cooperativity between two metal ions and a medium effect could result in excellent catalytic activities for
the cleavage of phosphate diesters.
artificial restriction enzymes for molecular biology which would
Introduction
be highly valuable in the manipulation of DNA, and developing
the anti-DNA drugs. Inspired by the structures of natural
nucleases, much work has focused on the synthesis of dinuclear
The development of synthetic agents able to hydrolytically
cleave phosphate diester bonds with high efficiency is a fascinat-
ing challenge and has received considerable interest during the
last two decades. These studies will provide several benefits,
such as shedding light on understanding of how catalysis works
under biological conditions and their potential applications as
8
–11
or polynuclear metal complexes.
Dinuclear system can con-
1
–7
tribute to the cleavage of a phosphate diesters in the following
four ways: (1) by double Lewis acid activation of a phosphate;
(2) through bifunctional catalysis in which the metal ions acti-
vate the phosphate and supply a metal-coordinated hydroxide
acting as nucleophile or base; (3) as an electrostatic reservoir of
positive charge to interact with the anionic phosphate to stabilize
the transition state for the phosphoryl transfer reaction; and (4)
by assisting the departure of the phosphate’s leaving group
through coordination.
a
College of Chemistry, Beijing Normal University, Xinjiekouwai Street
9, Beijing, 100875, China. E-mail: luzl@bnu.edu.cn;
1
Fax: +86-10-58801804; Tel: +86-10-58801804
b
State Key Laboratory of Applied Organic Chemistry, Lanzhou
University, Lanzhou, 730000, China
c
Nordion Inc., 447 March Road, Ottawa, ONK2K, 1X8, Canada
Recently, the Brown group has studied the systems comprising
mono- and dinuclear-Zn(II) complexes of 1,5,9-triazacycodode-
cane ([12]aneN , 1, Scheme 1) and 1,3-bis N -(1,5,9-triaza-
†
Electronic supplementary information (ESI) available: Tables of
kinetic data and method for triflate ion inhibition, plots of kinetics data
as a function of metal complexes concentrations and triflate, NMR and
mass spectroscopic data for the identity of ligands 5–9, and the inter-
mediates. See DOI: 10.1039/c2ob25624j
3
1
cyclododecyl)propane (di-[12]aneN , 2) in alcohols and found
3
that the dinuclear complex Zn -2 can promote the cleavage of a
2
7714 | Org. Biomol. Chem., 2012, 10, 7714–7720
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Experimental
Materials
Methanol (99.8% anhydrous), sodium methoxide (0.5 M solu-
tion in methanol), perchloric acid (70% aqueous solution), zinc
triflate, and tetrabutylammonium triflate were purchased from
Aldrich and used as supplied. 1,3-Dibromopropane, formal-
dehyde, formic acid, sodium hydroxide, concentrated hydro-
chloric acid, sodium borohydride, acetonitrile, ethanol, and
chloroform used in the experiments were purchased from Beijing
Chemical Reagents Company.
Scheme 1 Structures of mono-, di-[12]aneN
phosphate diesters 3–4.
3
ligands 1–2 and model
Synthesis
Syntheses of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP,
2
2
23
3
3
1
), methyl-p-nitrophenyl phosphate (MPNPP, 4), octahydro-
24
a,6a,9a-triazaphenalene (a precursor of [12]aneN , 10),
-methyl-1,5,9-triazacyclododecane (5),
triazacyclododecane (6), and 1,5,9-trimethyl-1,5,9-triazacyclo-
dodecane (7) were synthesized according to the literature pro-
3
25
1,5-dimethyl-1,5,9-
24
26
1
cedures and characterized by H-NMR.
Preparation of ligands 8 and 9
Ligand 8. To a solution of [12]aneN precursor 10 (3.22 g,
3
1
7.8 mmol) in 120 mL of dry acetonitrile was added dropwise
Scheme 2 Structures of N-methylated mono- and di-[12]aneN
ligands.
3
1,3-dibromopropane (1.79 g, 8.88 mmol) in 5 mL of dry aceto-
nitrile with stirring. The mixture was refluxed in an oil bath
maintained at 98 °C over 60 h to give a beige precipitate, which
was collected via filtration, washed with cold acetonitrile and
ether, and dried at room temperature under vacuum. This afforded
well-studied RNA model, namely 2-hydroxypropyl p-nitrophenyl
phosphate (HPNPP, 3) and DNA model methyl p-nitrophenyl
3
.7 g of the dibromide salts 11 with a yield of 74%. This product
was subsequently used as a precursor for ligands 8 and 9.
Compound 11 (1.46 g, 2.59 mmol) was dissolved in 100 mL
of absolute ethanol and placed in an ice-bath. Then 0.3 g
1
2–14
phosphate (MPNPP, 4) by a spectacular factor of up to 10
fold, when compared to the background reactions.
-
1
2,13
They
further explored almost every aspect of the catalytic cleavage of
the phosphate diesters with the dinuclear Zn(II) complexes of di-
(
7.9 mmol) of NaBH was slowly added to the above solution,
4
and the mixture was stirred overnight under nitrogen. After
quenching the reaction mixture with 1 M HCl, the solvent was
removed by rotary evaporation under reduced pressure. To the
residue was added 20 mL of 10 M NaOH solution and the
mixture was stirred at room temperature for 2 h. The free ligand
[
12]aneN ligands. In particular, they varied the structures of the
3
catalysts by using different metal ions and different linker struc-
tures (with various lengths and electronic properties), various
RNA and DNA model substrates, and different reaction
mediums (including methanol, ethanol, and aqueous
8
was extracted with chloroform, dried with sodium sulfate, and
14–21
ethanol).
Their work sheds light on the catalytic mechanism
finally evaporated under reduced pressure to afford the yellow oil
of the natural nucleases and provides rich information for prepar-
ing enzyme mimics. However, the steric effects of the catalyst
structures have not been investigated in detail, this also plays
very important roles in understanding reaction mechanisms and
controlling reactions.
Here we report on the preparation of a series of mononuclear
and dinuclear zinc(II) complexes with ligands 5–9 (Scheme 2),
which bear different numbers of N-methyl groups, and their cata-
lytic performance in the cleavage of phosphates 3 and 4 in
methanol. The incorporation of N-methyl groups in the [12]-
1
0
.94 g with a yield of 88%. H NMR (400 MHz, CDCl , ppm):
3
δ 2.66–2.64 (t, 8H), 2.45–2.38 (m 12H), 2.36–2.33 (m 4H),
13
2
.32–2.28 (m, 4H), 2.10 (s, 6H), 1.61–1.54 (m, 14H). C NMR
(
101 MHz, CDCl , ppm): δ 57.6, 54.3, 54.0, 51.6, 50.1, 49.1,
3
−1
4
3
1
1
5
8.8, 41.5, 25.2, 25.2, 24.8, 23.2. IR (KBr, cm ): 3304 (s),
227 (s), 2956 (s), 2687 (s), 2656 (s), 1612 (m), 1586 (w),
487 (m), 1464 (m), 1420 (m), 1307 (w), 1267 (w), 1140 (w),
089 (w), 1066 (w), 1015 (w), 923 (w), 745 (w), 668 (w),
78 (w). HR-MS (m/z) found (calcd) for C H N (M):
23 51 6
+
[
M + H] , 411.4172 (411.4175).
aneN backbone clearly reduced the catalytic activity of the cor-
3
responding metal complexes, especially the synergistic effect
Ligand 9. Compound 11 (2.03 g, 3.60 mmol) obtained via
between the two metal ions. The steric effects of N-methylations
the above process was added to 20 mL of a mixture of formal-
dehyde, hydrochloric acid, and formic acid (in a 10 : 1 : 10
volume ratio). After refluxing for 48 h and the evaporation of
in the [12]aneN ligands on the catalytic cleavage of phosphates
3
have been analyzed in detail in this work.
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water, the residue was mixed with 10 mL of concentrated HCl,
the resulted white precipitates was collected and washed with
cold ethanol and diethyl ether, and subsequently dried under
vacuum, which afforded the HCl salt of 9. The free ligand 9 was
obtained by base neutralization with 10 M NaOH, 1.34 g, yield
1
8
5%. H NMR (400 MHz, CDCl , ppm): δ 2.62 (m, 8H), 2.50
3
(
m, 16H), 2.36–2.32 (t, 4H), 2.25 (s, 12H), 1.68–1.61 (m, 14H).
1
3
C NMR (101 MHz, CDCl , ppm): δ 53.0, 52.9, 52.0, 49.2,
3
−1
4
1
1
9
4
3.2, 22.3, 22.1. IR (KBr, cm ): 3430 (s), 2959 (s), 2639 (s),
683 (m), 1633 (m), 1486 (s), 1461 (s), 1302 (w), 1257 (w),
227 (w), 1152 (w), 1111 (m), 1068 (m), 1047 (m), 1020 (m),
69 (w), 921 (m), 906 (m), 881 (w), 760 (m), 739 (m), 591 (w),
99 (w). HR-MS (m/z) found (calcd) for C H N (M):
2
5 55 6
Scheme 3 Synthesis of ligands 8 and 9.
+
[
M + H] , 439.4486 (439.4488).
Preparation of the stock solutions of the zinc(II) complexes
For the mononuclear complexes, the stock solutions were pre-
s
s
27
s
s
pared in situ at a pH equal to the corresponding pK in
a
methanol by addition of aliquots of stock solutions of sodium
methoxide, N-methylated-1,5,9-triazacyclododecanes (5–7) and
Zn(CF SO ) in a 0.5 : 1 : 1 molar ratio. No effects attributable
Scheme 4 Cleavage of HPNPP catalyzed by metal complexes of 5–9.
3
3 2
to the order of addition of the complex components were
observed in this case. The dinuclear complexes Zn -8 and Zn -9
Photophysics SX-17MV stopped-flow reaction analyzer thermo-
stated at 25.0 °C. Reactions were followed by monitoring the
rate of loss of the starting material at 280 nm and appearance of
the product p-nitrophenol at 320 nm. The study of the base
2
2
were prepared as a 2.5 mM solution in methanol according to
12
the procedure established in the previous literatures. This
involved the sequential addition of aliquots of stock solutions of
dependence of the cleavage of HPNPP catalyzed by Zn -8 was
2
sodium methoxide, dinuclear ligand (8 or 9), and Zn(CF SO )
3
3 2
performed by “pH jump” experiments where one syringe of the
stopped-flow reaction analyzer contained a 0.4 mM solution of
such that the relative amounts were of a 1 : 1 : 2 molar ratios,
respectively. The solution being kept for 50 min before the
kinetic measurement were recorded in order to ensure that the
dinuclear complexes had fully formed.
Zn -8 and another syringe contained a 0.08 mM solution of
HPNPP in methanol along with the required amount of perchlo-
ric acid or sodium methoxide.
2
Kinetics
Results and discussion
The rates of cleavage of HPNPP (0.04 mM) catalyzed by the
mononuclear complexes Zn-L (L = 5–7) and dinuclear complex
Zn -8 and Zn -9 as well as that of methanolysis of MPNPP cata-
Synthesis of ligands 8 and 9
The synthesis of new ligands 8 and 9 was straightforward. The
preparation began with the alkylation reactions between the
2
2
lyzed by complexes Zn-5 and Zn -8 were followed by monitor-
2
[
12]aneN precursor 10 and 1,3-dibromopropane (Scheme 3),
ing the appearance of p-nitrophenol at 320 nm using a Cary 300
UV-vis spectrophotometer with the cell compartment thermo-
stated at 25.0 ± 0.1 °C. Second order rate constants for the meth-
oxide reactions of 3 and 4 were taken from literature as (2.56 ±
3
which produced bicyclic amidinium salts 11 as the key inter-
mediates. The reduction of the intermediate 11 with NaBH4,
further acid hydrolysis and base neutralization yielded ligand 8,
0
2
.16) × 10−
5 °C.
3
M
−1 −122
s
and (7.9 ± 0.6) × 10
−7
M
−1 −128
s
at
which contains di-N-methyl groups. Through the Eschweiler–
Clarke reaction, e.g., the reaction of 11 with a mixture of formal-
dehyde and formic acid, with subsequent acid hydrolysis and
neutralization, tetra-N-methylated ligand 9 was obtained in good
yield. The two new ligands were fully characterized with
The dependence of the methanolysis rate on base concen-
tration of methyl p-nitrophenyl phosphate (MPNPP, 4) catalyzed
by Zn -8 was studied by adding aliquots of stock solutions of
2
1
13
H-NMR, C-NMR, IR and HR-MS (see ESI†).
sodium methoxide or perchloric acid to the cell containing
1
In the H-NMR spectra, the protons from methylene groups of
0
.4 mM of the [Zn -8] complex. The reaction was initiated
2
[
12]aneN backbone appear around 1.60, 2.50, and 2.80 ppm as
immediately by the addition of MPNPP solution so that the final
substrate concentration in the UV cell was 4 × 10− M. In
general, the reactions followed good first-order kinetics up to
about three half-times of the methanolysis reaction. The pseudo-
first order rate constants (kobs) were determined by an NLLSQ
fitting of the absorbance versus time traces to a standard expo-
nential model.
3
5
multiplets, which are consistent with those reported in the litera-
29
tures. The N-methyl groups on the [12]aneN unit appear as
3
24
singlet at 2.06 and 2.25 ppm for ligand 8 and 9, respectively.
Catalytic cleavage of HPNPP
The rates of the cleavage of HPNPP (0.04 mM) catalyzed by
The catalytic activities of the metal complexes with ligands 5–9
the Zn -8 complex were determined using an Applied
on the cleavage of HPNPP (Scheme 4) were investigated in
2
7716 | Org. Biomol. Chem., 2012, 10, 7714–7720
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methanol. To avoid the effect of triflate counter ions for the fast
the k values are also similar to those of zinc(II) complexes of
2
reactions with Zn -8, the kinetic data were corrected for the
triflate inhibition constants (see ESI†).
[12]aneN3 ligands containing N-benzyl or N-(3-benzyl-1,2,3-
triazolyl)methyl moieties in our previous work.
2
30
(
i) Cleavage of 3 with mononuclear zinc(II) complexes. Fig. 1
(
ii) Cleavage of 3 with dinuclear zinc(II) complexes. In the
shows the plots of the observed pseudo-first order rate constants
for the cleavage of HPNPP 3 as a function of [Zn-L] (L = 5, 6,
) in the presence of 0.5 equivalent of CH O to set the pH of
3 s
the solution at self-buffered conditions.
For ligands 5 and 6, their plots show upward curvatures, indi-
cating a bimolecular process which are similar to that of mono-
nuclear complexes with [12]aneN ligands in the literatures.
By fitting the plots in the eqn (1), it affords k2 and k3 for
Zn-5 and Zn-6, respectively.
preliminary work, the reaction conditions were optimized by
varying equivalents of base (sodium methoxide), zinc(II) ion,
and by adjusting the mixing time. The same conclusion was
reached as the literature reported:
were obtained in situ through sequential addition of stock sol-
utions of sodium methoxide, ligand, and Zn(OTf) to anhydrous
methanol such that [CH O ] : [8/9] : [Zn(II)] = 1 : 1 : 2.
The dinuclear complex Zn -8 with one equivalent of added
methoxide produces a catalyst for the cleavage of 3, which has
remarkable activity in methanol. Fig. 2 includes a plot of kobs vs.
CH O ] = [Zn -8] determined at a measured pH of 9.2 ± 0.1.
The rapid rate of the reaction requires it to be monitored by
stopped-flow spectrophotometry at 320 nm, which is the wave-
length corresponding to the appearance of p-nitrophenol. After
correction for the triflate inhibition, the plot of kobs
Zn -8] is linear, the second order rate constant (k ) evaluated
from the gradient of the linear plot is (3.03 ± 0.098) × 10 M s .
It can be seen that the k value exhibited by Zn -8 is 1.18 × 10 -
−
s
7
12,13
the most reactive catalysts
12
2
3
−
obs
obs
3
2
obs
obs
2
kobs ¼ k2 ½Zn ꢀ Lꢁ þ k3 ½Zn ꢀ Lꢁ
ð1Þ
−
s
3 2 s
[
For Zn-7, the plot is almost linearity, indicating that a bimole-
cular process is prevented due to the steric effect from the three
N-methyl groups in the ligand backbone.
All of the kinetic data is listed in Table 1 for comparison. It
can be seen that N-methylations of the [12]aneN unit gradually
corr
versus
3
[
2
2
reduced the catalytic activity of their corresponding mononuclear
species. These reductions were not too great, since the k values
3
−1 −1
2
6
2
2
are very close for all three mononuclear complexes. However,
the incorporation of N-methyl groups greatly affects the synergis-
tic effect among the mononuclear species. Zinc(II) complexes
with ligands bearing mono- or di-N-methyl groups (5 and 6,
respectively) can still associate to bring bimolecular process.
Meanwhile, the presence of tri-N-methyl groups in the [12]aneN3
unit resulted in no synergetic effect at all. It is noteworthy that
fold greater than that for the methoxide-promoted cleavage of 3
OMe
−3
−1 −1
(
k2
= 2.56 × 10
M
s ). The synergetic effect between
two [12]aneN units is 561-fold when compared to those of Zn-
3
5
and Zn-6.
For the reaction of 3 with Zn -9, the kinetic data show that the
2
rate constants (kobs) versus concentration exhibit a linear relation-
−1 −1
ship. The corresponding k value of (4.35 ± 0.06) M
s
for
2
Fig. 1 Plots of kobs vs. [Zn-L] (L = 5 ●, 6 ■, 7 ▲) for the cleavage of
2
Fig. 2 Plots of kobs vs. [Zn -L] (L = 8 ▲, 9 ●) for the cleavage of
−5
s
−5
−
HPNPP (4 × 10 M) at [–OCH
3
]/[Zn(II)] = 0.5,
s
pH = 9.7 ± 0.1 for Zn-
HPNPP (4 × 10 M) in the presence of 1 eq. of added CH
complex giving pH = 9.2 ± 0.1 for Zn -8 (left Y axis) and pH = 9.4 ±
0.1 for Zn -9 (right Y axis), at 25 ± 0.1 °C.
3
O
per
s
s
s
s 2 s
s
5
,
s
pH = 9.5 ± 0.1 for Zn-6, and
s
pH = 9.4 ± 0.1 for Zn-7 at 25.0 ±
0
.1 °C.
2
Table 1 Kinetic constants for the cleavage of 3 mediated by zinc(II) complexes of ligand 5–9 at 25.0 ± 0.1 °C
Catalytic acceleration at
self-buffered pH
s
s
s
(M−1 s−1
× 10−3 (M−2 s−1
s
Cat.
pH
k
2
)
k
3
)
Synergetic effect
−OMe
Zn-1
Zn-5
Zn-6
Zn-7
—
2.56 × 10
18.9
2.7 ± 0.1
−3a
—
1.8 ± 0.4
0.71 ± 0.03
a
a
8
9.1 ± 0.1
9.7 ± 0.1
9.5 ± 0.1
9.4 ± 0.1
9.5 ± 0.1
9.2 ± 0.1
9.4 ± 0.1
3.4 × 10
7
1.2 × 10
7
2.70 ± 0.08
1.88 ± 0.08
(2.75 ± 0.10) × 103
(3.03 ± 0.08) × 10
4.35 ± 0.06
0.20 ± 0.02
2.0 × 10
7
—
—
—
—
1.7 × 10
5a
12
Zn
2
Zn
2
Zn
2
-2
-8
-9
7230
561
1.1
2.0 × 10
10
4.4 × 10
7
4.0 × 10
a
See ref. 12.
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this reaction is almost double that of Zn-7, indicating that there
is no significant synergetic effect between the two metal ions.
shows some evidence of saturation binding, similar to the cataly-
tic behavior of Zn -2. However, after correction of the effect of
2
the weaker triflate inhibition, the plot becomes almost linear,
which is difficult to fit with a standard Michaelis–Menten kin-
Catalytic cleavage of MPNPP
etics as for that of Zn -2. The linear fit of the plot afforded a
2
As a DNA model phosphate, the cleavage of MPNPP 4 is much
slower when it is compared with the cleavage of 3. In the case of
−1
−1 −1
second rate constant as (1.78 ± 0.04) × 10
M
s
, which is
5
2
.2 × 10 -fold greater than that of methoxide-promoted cleavage
3
, the cleavage of the p-nirophenolate group is actually an intra-
of 4, the synergetic effect between the [12]aneN3 units is
approximately 217-fold when compared with that of Zn-5. In
comparison with Zn -2, N-methylations in complex Zn -8
3
molecular ring closure which is more than 10 -fold faster than
17
that of 4. A nucleophile species such as hydroxide or alkoxide
is not directly involved in the cleavage of 3. Meanwhile in the
case of 4, the leaving group p-nitrophenole is replaced by a
nucleophile through intermolecular or catalyst-mediated attack.
In order to obtain information about the steric effect on the clea-
vage of 4, the kinetic data were measured in the presence of
2
2
clearly reduced the catalytic activity by approximately 620-fold
in terms of the k values (see Table 2).
2
In the multi-step mechanism of the catalytic cleavage of DNA
model 4, the rate determining step was proposed to be the third
13
step, i.e., the chemical cleavage of 4-nitrophenole from 4,
metal complexes Zn-5 and Zn -8.
2
which often resulted in the saturation kinetics. The different con-
centration dependencies shown by Zn -8 (linear behavior) and
(
i) Cleavage of 4 with Zn-5. The methanolysis of 4 catalyzed
2
by Zn -2 (saturation behavior) may result from the weaker
by Zn-5is very slow. The rate constants of the reaction were
obtained from the initial rate procedure by fitting the first 5-10%
of the absolute versus time traces by linear regression and then
converting to first order rate constants (kobs) by dividing them by
the expected absorbance change, if the reaction were to reach
2
binding strength between 4 and Zn -8 due to the steric effect
2
from the N-methyl groups, which suggests that the saturation
binding was not reached in the studied concentration range of
Zn -8. For this reason, pH dependencies of the kinetics in the
2
1
7
cleavage of both phosphates catalyzed by Zn -8 were investi-
1
00% completion, as reported in the literature. The plot of the
kobs versus [Zn-5] is shown in Fig. 3A, which has linearity
2
gated, which supports the known mechanism.
−4
dependence. The second rate constant is k = (4.1 ± 0.2) × 10
2
−
1
−1
M
s
, corresponding to an enhancement of approximately
2
pH jump experiments for the reactions of 3 and 4 with Zn -8
5
20-fold, when compared to that of methoxide.
In the cleavage of 3 and 4 with Zn -2 in methanol, the reactions
2
(
ii) Cleavage of 4 with Zn -8. For the methanolysis of 4 with
follow the same mechanism, which includes two binding steps
and one chemical cleavage step. However, the rate-limiting step
with 3 is its binding process while that with the slower reacting
2
Zn -8, the kinetics were measured by monitoring the entire
2
process using UV spectroscopy. The plot of kobs versus [Zn -8]
2
4
is the chemical step of cleavage of the bound substrate to
release p-nitrophenol. These have been clearly revealed by their
kinetics of the concentration dependencies. In the case of Zn -8,
2
the kinetics of the concentration dependence for the reactions
with 3 and 4 are both in linearity, making it difficulty to clarify
the rate-limiting step. Thus the influence of pH on the reactions
was checked by varying the base amount in the catalytic system
by using the pH-jump procedure. The kinetic data are plotted in
Fig. 4. It can be seen that the kobs for the cyclization of 3
decreased as the methoxide ratio was increased, but it increased
as the quantity of base was reduced. The methanolysis of 4
s
shows a bell-shaped pH/rate profile.
The results are completely consistent with those found for the
Fig. 3 (A) Plot of kobs vs. [Zn-5] for the cleavage of MPNPP (4 × 10−5 M)
s
s
at
s
pH 9.8 ± 0.1 and 25 ± 0.1 °C. (B) Plot of kobs vs. [Zn
2
-8] for the clea-
−5
s
Zn -2 system (see Scheme 5). For the reaction with HPNPP 3,
2
vage of MPNPP (4 × 10 M) at
s
pH 9.1 ± 0.1 and 25 ± 0.1 °C. The
the increase in rate with added acid is consistent with a process
depending on binding the substrate to the complex devoid of an
associate methoxide with its higher net positive charge attracting
dotted line is presented as a visual aid directed through all actual data
points (■); the solid line is a linear fit through the data corrected for inhi-
bition by the triflate counterions (▲).
Table 2 Kinetic data for the cleavage of 4 mediated by Zn-5 and Zn
2
-8 at 25.0 ± 0.1 °C
Catalytic acceleration at
self-buffered pH
s
s
s
(M−1 s−1
s
Cat
pH
k
2
)
Synergetic effect
−OMe
Zn-5
—
(7.9 ± 0.6) × 10−
(4.1 ± 0.2) × 10
110 ± 27
−7a
—
6.0 × 10
2.6 × 10
4
6
9.7 ± 0.1
9.5 ± 0.1
9.2 ± 0.1
b
12
Zn
Zn
2
-2
-8
—
217
(1.78 ± 0.04) × 10−
1
8.3 × 10
9
2
a
b
See ref. 28. See ref. 12.
7718 | Org. Biomol. Chem., 2012, 10, 7714–7720
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Fig. 4 Plots of the observed pseudo-first order rate constants for the
−5
methanolysis of 4 × 10 M HPNPP (▲, left axis) catalyzed by 3.2 ×
−
4
−5
1
1
0
0
0
M Zn
M Zn
2
-8 or 4 × 10 M MPNPP (●, right axis) catalyzed by 1 ×
Scheme 6 Structures of ligand 13–15.
−3
−
2
-8 as a function of the [CH
3
O ]/[ Zn
2
-8] ratio at 25 ±
.1 °C.
constant for the cleavage of 3 by Zn -8, is reduced by 91-fold.
2
Meanwhile the synergetic effect between the two [12]aneN3
units is reduced by 13-fold. For the reactions with 4 catalyzed by
Zn -8, it was found that the second order rate constant is reduced
2
by 620-fold relative to that of Zn -2.
2
The electronic effect caused by the oxyanion bridging group
in Zn -12 (Scheme 6) reduced the second order rate constant for
2
−1
−1
the cleavage of HPNPP to be 7.6 M
700-fold rate reduction. In contrast, the presence of di-N-
s
and thus caused a
19
3
Scheme 5 Proposed mechanism for the cleavage of 3/4 promoted by
Zn -8.
methyl groups in Zn -8 caused only a 91-fold reduction of
2
2
activity. However, the presence of tetra-N-methyls in Zn -9
2
resulted in 63 000 times less activity in terms of the k value.
2
the negatively charged HPNPP. The chemical step of methoxide-
Thus the steric effects in the di-[12]aneN systems acuminated
3
dependent cyclization (k ) is faster than the rearrangement step
3
exponentially.
(
k ). For the reaction with MPNPP 4, the maximum rate occurs
2
−
Another noteworthy point is that the steric effect has to be
analyzed carefully. It was found that the addition of methyl sub-
stituents in ligands 13–15 (Scheme 6) provided a rate enhance-
ment in comparison with those of the non-substituted ligands for
when the [CH O ]/[Zn -8] ratio is unity, which clearly indicates
3
2
that the process requires the combination of substrate binding to
Zn(II) ion and a basic role involving methoxide. Thus the rate
limiting step in the catalytic cleavage of HPNPP is the binding
process, and that for slow reacting MPNPP is the chemical clea-
vage of the P–O bond.
31,32
the cleavage of 3.
For mononuclear zinc(II) complexes of
1
3a and 13b, a factor of 4 rate enhancement was observed at the
self-buffered conditions for methylated ligand 13b. In the dinuc-
3
lear systems, complex Zn -14b was 10 times more reactive than
2
Analysis of the steric effect on the catalytic cleavage of
phosphates
complex Zn -14a, and complex Zn -15b was 10 times more
2
2
reactive than Zn -15a. These enhancements have been attributed
2
to the decrease of local polarity due to the hydrophobic property
of methyl group. In the case of ligand 5–9, the N-methylations
directly affect on the coordinating nitrogen donor, which clearly
weakens the ligand binding to the metal ions as well as the
complex binding to the substrate, both aspects result in poor
catalysis.
It is apparent that the incorporation of N-methyl groups in the
[
12]aneN backbone clearly impairs the catalytic efficiency in
3
the cleavage of RNA and DNA model phosphates 3 and 4.
However, the extent of this effect varies among the different
reaction systems.
For the cleavage of 3 with Zn:L (L = 5, 6, 7), the N-methyla-
Nevertheless, Zn -8 is still among the most active catalysts for
2
tions has little effect on the second order rate constant k values
2
−1
−1
the cleavage of the DNA and RNA model phosphates. The
second order rate constants in the methanolysis of HPNPP and
as they are 2.7, 2.7, and 1.88 M
s , respectively, for the three
mononuclear complexes. However, their association ability to
form the cooperative dinuclear species was greatly decreased
along the increase of the number of N-methyl groups due to the
6
5
MPNPP are respectively 1.18 × 10 -fold and 2.2 × 10 -fold
greater than those of methoxide-promoted reactions. The syner-
getic effect in the cleavage of HPNPP with Zn -8 is 561-fold. In
2
steric effect. The values of k are decreased by 2.1 and 9-fold
3
the presence of 1 mM of Zn -8 at self-buffered conditions, the
2
when one and two N-methyls are incorporated into [12]aneN3.
For the complex containing fully N-methylated ligand (Zn-7),
there is almost no upward curvature in the plot of kobs vs. [Zn].
In comparison with the reactivity of methoxide, the activities of
these mononuclear complexes are still significant, their second
order rate constants are approximately 1000 times higher than
that of methoxide.
methanolysis of HPNPP and MPNPP can be accelerated by
10
9
4
.4 × 10 -fold and 8.4 × 10 -fold, respectively.
Conclusions
The present work has systematically investigated the cleavage of
RNA and DNA model phosphates HPNPP and MPNPP cata-
lyzed by zinc(II) complexes of the N-methylated mono- and
For the dinuclear complexes, the steric effects are more
obvious. Compared to those of Zn -2, the second order rate
2
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Org. Biomol. Chem., 2012, 10, 7714–7720 | 7719

View Article Online
di-[12]aneN ligands (5–9). The obtained results revealed that
5 J. Weston, Chem. Rev., 2005, 105, 2151.
3
6
7
R. Wolfenden and M. J. Snider, Acc. Chem. Res., 2001, 34, 938.
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N-methylation of the [12]aneN backbone greatly reduced the
3
cooperative behavior among the mononuclear Zn(II) complexes,
a common phenomenon in a reduced dielectric alcohol medium.
Such cooperative action would result in the synergetic effect and
significant catalytic activity. The activities of the dinuclear zinc(II)
8 C. Liu and L. Wang, Dalton Trans., 2009, 227.
F. Mancin and P. Tecilla, New J. Chem., 2007, 31, 800.
9
10 R. S. Brown, Z.-L. Lu, C. T. Liu, W. Y. Tsang, D. R. Edwards and
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11 D. E. Wilcox, Chem. Rev., 1996, 96, 2435.
complexes for the di-[12]aneN ligands containing di-N-methyl
3
(
8) and tetra-N-methyl (9) substituents for cleaving HPNPP have
12 A. A. Neverov, Z.-L. Lu, C. I. Maxwell, M. F. Mohamed, C. J. White,
J. S. W. Tsang and R. S. Brown, J. Am. Chem. Soc., 2006, 128, 16398.
been impaired by 91-fold and 63 000-fold, respectively. The
reduction of the activity in the cleavage of MPNPP follows a
similar trend. The kinetic results indicate that the steric effects
derived directly from the coordinating nitrogen donors greatly
reduced the binding activation process and accumulated expo-
nentially. Nevertheless, some zinc(II) complexes of N-methylated
mono- and di-[12]aneN ligands still provide significant catalytic
efficiency in methanol. Dinuclear complex Zn : 8 can accelerate
the cleavage of each phosphate by a factor of 10
tive to the background reactions at concentration of 1 mM and
self-buffered conditions. The synergetic effects between the two
13 Z.-L. Lu, C. T. Liu, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc.,
2007, 129, 11642.
14 S. E. Bunn, C. T. Liu, Z.-L. Lu, A. A. Neverov and R. S. Brown, J. Am.
Chem. Soc., 2007, 129, 16238.
1
1
1
1
5 C. T. Liu, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc., 2008,
130, 16711.
6 C. T. Liu, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc., 2008,
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3
2
9
–10
folds rela-
19 M. F. Mohamed, A. A. Neverov and R. S. Brown, Inorg. Chem., 2009,
8, 11425.
4
[
12]aneN units of Zn -8 are 561 and 340 folds, respectively, for
3 2
2
0 W. Y. Tsang, D. R. Edwards, S. A. Melnychuk, C. T. Liu, C. Liu,
A. A. Neverov, N. H. Williams and R. S. Brown, J. Am. Chem. Soc.,
2009, 131, 4159.
the catalytic cleavage of the two phosphates. Results from this
work have provided the insight into the steric effects on reactions
catalyzed by artificial nucleases and further confirmed that a
combination of the cooperative effect between two metal ions
and a solvent effect can lead to significant catalytic activities for
the cleavage of phosphate diesters.
21 D. R. Edwards, W.-Y. Tsang, A. A. Neverov and R. S. Brown, Org.
Biomol. Chem., 2010, 8, 822.
22 J. S. W. Tsang, A. A. Neverov and R. S. Brown, J. Am. Chem. Soc.,
2003, 125, 1559.
23 A. J. Kirby and M. Younas, J. Chem. Soc. B, 1970, 1165.
24 R. W. Alder, R. W. Mowlam, D. J. Vachon and G. R. Weisman, J. Chem.
Soc., Chem. Commun., 1992, 507.
2
2
5 J. Notni, H. Goerls and E. Anders, Eur. J. Inorg. Chem., 2006, 1444.
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Acknowledgements
3
2, 7755.
The authors gratefully acknowledge the financial assistance from
the Program for New Century Excellent Talents at Universities
2
7 For the designation of pHin non-aqueous solvents we use the forms rec-
ommended by the IUPAC, Compendium of Analytical Nomenclature.
Definitive Rules 1997 3rd edn, Blackwell, Oxford, U.K. 1998. If one cali-
brates the measuring electrode with aqueous buffers and then measures
the pH of an aqueous buffer solution, the term
(NCET-08-0054), the Ministry of Education of China; Nature
Science Foundation of China (20972019); the Fundamental
Research Funds for the Central Universities (2009SC-1), Beijing
Municipal Commission of Education, and the National Student
Innovation Training Program (SITP) for undergraduates.
w
w
pH is used; if the elec-
trode is calibrated in water and the ‘pH’ of the neat buffered methanol
solution then measured, the term
s
w
pH is used; and if the electrode is cali-
brated in the same solvent and the ‘pH’ reading is made, then the term
s
s
−16.77
pH is used. Since the autoprotolysis constant of methanol is 10
,
s
neutral
s
pH is 8.4
2
2
8 A. A. Neverov and R. S. Brown, Inorg. Chem., 2001, 40, 3588.
9 J. Zan, H. Yan, Z.-f. Guo and Z.-L. Lu, Inorg. Chem. Commun., 2010,
13, 1054.
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31 M. F. Mohamed and R. S. Brown, J. Org. Chem., 2010, 75, 8471.
32 M. F. Mohamed, I. Sanchez-Lombardo, A. A. Neverov and R. S. Brown,
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39.
7720 | Org. Biomol. Chem., 2012, 10, 7714–7720
This journal is © The Royal Society of Chemistry 2012